Nanopore Sensor Calibration and Operation With A Fluidic Passage

ABSTRACT

A first fluidic solution having a first ionic concentration is provided in a first fluidic reservoir in direct fluidic connection with a nanopore. A second fluidic solution, having a second ionic concentration, is provided in a second fluidic reservoir disposed in fluidic connection with the nanopore through a fluidic passage having at least one fluidic section in which the section length is greater than the section width. The electrical potential local to the fluidic passage is measured, and the resistance of both the fluidic passage the nanopore are determined based on the fluidic passage electrical potential. The fluidic passage resistance is compared with the nanopore resistance and at least one of the first and second ionic concentrations is adjusted based on the comparison. The measuring, determining, comparing, and adjusting steps are conducted until the fluidic passage resistance is between about 0.1 times and about 10 times the nanopore resistance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/015,277, filed Feb. 4, 2016, which claims the benefit of U.S.Provisional Application No. 62/112,630, filed Feb. 5, 2015, the entiretyof both of which are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Contract No.5DP10D003900 awarded by the NIH. The Government has certain rights inthe invention.

BACKGROUND

This invention relates generally to sensing systems that employ ananopore sensor, and more particularly relates to techniques for sensingspecies as the species translocate a nanopore sensor.

Both solid-state nanopores and biological nanopores are increasingly thefocus of considerable effort in the development of a low cost, highthroughput sensing system that can be employed for sensing a wide rangeof species, including single molecules such as polymer molecules. Acommon approach in nanopore-based sensing employs the measurement ofionic current flow through a nanopore that is provided in a highlyresistive amphiphilic membrane between electrodes provided on eitherside of the membrane. As a molecule such as a polymer analyte like DNAis caused to translocate the nanopore, the ionic current flow throughthe nanopore is modulated by the different nucleotide bases of a DNAstrand. Measurement in changes in ionic current flow can be carried outin order to determine a sequence characteristic of the polymer strand.Nanopore devices for detection of analytes other than polynucleotideshave also been reported, for example in International Patent ApplicationPCT/US2013/026414, published as WO2013/123379, for the detection ofproteins. Whilst there has also been considerable effort in developingmethods and systems using solid state nanopores in order to sequenceDNA, there remain a host of challenges for commercial realization. Inaddition, various configurations of nanopores pose particularchallenges. For example, in the use of an array of nanopores in whichionic current flow through each nanopore in the array can be measured, ameasurement can be made between a common electrode and a plurality ofelectrodes provided on the respective opposite side of each nanopore.Here the plurality of electrodes needs to be electrically isolated fromeach other, limiting the level of integration density of nanoporedevices.

Biological nanopores in some respects are advantageous over solid statenanopores in that they provide a constant and reproducible physicalaperture. However the amphiphilic membranes in which they are providedare in general fragile and may be subject to degradation, providingionic leakage pathways through the membrane. The speed of translocationof an analyte through a biological nanopore can be controlled by the useof an enzyme. Enzyme-assisted translocation of polynucleotides istypically on the order of 30 bases/second. In order to increase thethroughput rate of analyte, much higher translocation speeds aredesirable, but it is found that in general, the measurement of the asensing signal can be problematic.

In order to circumvent the technical challenges posed by the ioniccurrent measurement method for nanopore sensing, several alternativenanopore sensing methods have been proposed. Such alternative methodsare in general directed to an arrangement in which there is recordedrelatively local nanopore signals employing electronic sensors that areintegrated with the nanopore. These nanopore sensing methods include,e.g., measurement of capacitive coupling across a nanopore andtunnelling current measurements through a species translocating ananopore. While providing interesting alternative sensing techniques,such capacitive coupling and tunnelling current measurement techniqueshave not yet improved upon the conventional ionic current detectiontechnique for nanopore sensing, and ionic current detection techniquesremain challenged by signal amplitude and signal bandwidth issues.

SUMMARY OF THE INVENTION

There is provided herein a methodology for calibrating and operating ananopore sensor that overcomes the historical limitations ofconventional nanopore sensors by measuring the local electricalpotential at a fluidic passage provided in the sensor. In the method, afirst fluidic solution having a first ionic concentration is provided ina first fluidic reservoir that is in direct fluidic connection with ananopore. A second fluidic solution, having a second ionicconcentration, is provided in a second fluidic reservoir. The secondfluidic reservoir is disposed in fluidic connection with the nanoporethrough a fluidic passage having at least one fluidic section in which alength of the fluidic section is greater than a width of the fluidicsection.

To calibrate the nanopore sensor, the electrical potential local to thefluidic passage is measured, and the fluidic passage resistance and thenanopore resistance are determined based on the measured fluidic passageelectrical potential. The fluidic passage resistance is compared withthe nanopore resistance and at least one of the first ionicconcentration and the second ionic concentration is adjusted based onthe comparison. The steps of measuring, determining, comparing, andadjusting are conducted until the fluidic passage resistance is betweenabout 0.1 times and about 10 times the nanopore resistance.

In operation, there is provided in the first fluidic solution at leastone molecule. The molecule is translocated from the first fluidicsolution to the second fluidic solution through the nanopore and thefluidic passage. During the translocation of the molecule, atranslocation electrical potential local to the fluidic passage ismeasured.

This nanopore sensor calibration and operation enables high sensitivityand large bandwidth, with a localized, large sensing signal that isproportional to nanopore ionic current. As a result, nanopore sensingapplications such as DNA sequencing can be accomplished with thenanopore sensor at a very high integration density and throughput ofanalyte. Other features and advantages of the invention will be apparentfrom the following description and accompanying figures, and from theclaims

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic circuit diagram of a first example nanoporesensor configuration for measuring local electrical potential;

FIG. 1B is a circuit diagram of an example transistor implementation ofthe nanopore sensor configuration of FIG. 1A;

FIG. 1C is a schematic circuit diagram of a second example nanoporesensor configuration for measuring a local electrical potential;

FIG. 1D is circuit diagram of an example transistor implementation ofthe nanopore sensor configuration of FIG. 1C;

FIG. 1E is a circuit diagram of an example transistor implementation ofa combination of the sensor configurations of FIGS. 1A and 1C;

FIG. 1F is a schematic plan view of a single electron transistorimplementation of a nanopore sensor configuration for measuring localelectrical potential;

FIG. 1G is a schematic plan view of a quantum point contactimplementation of a nanopore sensor configuration for measuring localelectrical potential;

FIG. 1H is a schematic side view of a lipid bilayer includingfluorescent dye arranged for implementation of a protein nanopore sensorconfiguration for measuring local electrical potential;

FIG. 2A is a schematic diagram and corresponding circuit elements for ananopore sensor configuration for measuring local electrical potential;

FIG. 2B is a circuit diagram for the nanopore sensor transistorimplementation of FIG. 1B;

FIG. 3A is a schematic side view of the geometric features of a nanoporesensor configuration for measuring local electrical potential as-definedfor quantitative analysis of the sensor;

FIGS. 3B-3C are plots of the electrical potential in a nanopore of ananopore sensor for measuring local electrical potential, here plottedas a function of distance from the nanopore into the cis reservoir, fora configuration in which the cis and trans reservoirs include fluidicsolutions of equal ionic concentration and for a configuration in whichthe cis and trans reservoirs include fluidic solutions of unequal ionicconcentration, respectively;

FIGS. 3D-3E are plots of the electrical field in a nanopore of ananopore sensor for measuring local electrical potential, correspondingto the plots of electrical potential of FIGS. 3A-3B, respectively;

FIG. 4A is a plot of the change in potential in a nanopore for a 50nm-thick nanopore membrane and a configuration of a 1 V transmembranevoltage (TMV) for electrophoretic species translocation as a dsDNAmolecule translocates through the nanopore, as a function of theC_(Cis)/C_(Trans) ionic concentration ratio for various nanoporediameters below 10 nm where the nanopore is configured for localelectrical potential measurement;

FIG. 4B is a plot of the change in potential in the trans reservoir fora 10 nm-diameter nanopore at 1 V TMV for the conditions of the plot ofFIG. 4A;

FIG. 4C is a plot of noise sources and signal as a function of recordingbandwidth for a nanopore sensor configured for local electricalpotential measurement;

FIG. 4D is a plot of the bandwidth of a nanopore sensor configured forlocal electrical potential measurement as a function of cis chambersolution concentration for a range of reservoir solution concentrationratios;

FIG. 4E is a plot of signal decal length from the nanopore site in ananopore configured for local electrical potential measurement as afunction of cis and trans reservoir solution concentration ratio;

FIG. 5 is a schematic view of a nanopore sensor including a fluidicpassage connected between a first reservoir, here the trans reservoir,and a nanopore in a support structure;

FIG. 6 is a circuit model of the of nanopore sensor of FIG. 5;

FIG. 7 is a plot of the ratio of measured transconductance signal tomaximum achievable transconductance signal as a function of the ratiobetween the fluidic passage resistance and the nanopore resistance;

FIG. 8 is a schematic side view of the nanopore sensor of FIG. 5 withthe definition of geometric parameters;

FIG. 9 is a plot of the ratio of resistance of the fluidic passage toresistance of the nanopore in the sensor of FIG. 5 as a function ofratio of fluidic passage diameter to fluidic passage length, forselected sensor dimensions;

FIG. 10 is a plot of the of the ratio of measured transconductancesignal to maximum achievable transconductance signal as a function ofthe ratio fluidic passage diameter to fluidic passage length, forselected sensor dimensions;

FIG. 11 is a schematic side view of a first fluidic passageconfiguration;

FIG. 12 is a schematic side view of a fluidic passage disposed on asupport structure for a nanopore;

FIG. 13 is a schematic side view of a anodized aluminum oxide fluidicpassage configuration;

FIG. 14 is a schematic side view of a lateral fluidic passageconfiguration;

FIGS. 15A-15B are schematic top-down cross sections of example lateralfluidic passage configurations;

FIGS. 16A-16E are schematic side views of fluidic passage configurationsarranged with elements for making a local electrical potentialmeasurement.

FIG. 17 is a schematic view of a nanopore sensor configured for localelectrical potential measurement with a nanowire FET disposed on amembrane;

FIG. 18 is a perspective view of one example implementation of thenanopore sensor configuration of FIG. 17;

FIGS. 19A-19B are a schematic view of a nanopore sensor configured forlocal electrical potential measurement with a nanowire FET disposed on agraphene membrane, and a plan view of an example implementation of thisnanopore sensor, respectively;

FIGS. 20A-20B are a schematic view of a nanopore sensor configured forlocal electrical potential measurement with a graphene layer disposed ona nanowire FET, and a plan view of an example implementation of thisnanopore sensor, respectively;

FIGS. 21A-21B are a schematic view of a nanopore sensor configured forlocal electrical potential measurement with a graphene membrane, and aplan view of an example implementation of this nanopore sensor,respectively;

FIGS. 22A-22D are schematic plan views of example locations of ananopore with respect to a nanowire in a nanopore sensor configured forlocal electrical potential measurement;

FIG. 23 is a plot of the sensitivity of a nanowire in a nanopore sensorconfigured for local electrical potential measurement before and afterformation of a nanopore at the nanowire location;

FIG. 24A is a plot of i) measured ionic current through a nanopore andii) measured nanowire FET conductance, respectively, as DNA translocatesthrough a nanopore in a nanopore sensor configured for local electricalpotential measurement, for a TMV of 2 V and 100:1 cis/trans reservoirsolution concentration ratio, with a local potential measurement made inthe trans reservoir;

FIG. 24B is a plot of i) measured ionic current through a nanopore andii) measured nanowire FET conductance, respectively, as DNA translocatesthrough a nanopore in a nanopore sensor configured for local electricalpotential measurement, for a TMV of 2.4 V and 100:1 cis/trans reservoirsolution concentration ratio, with a local potential measurement made inthe trans reservoir;

FIG. 24C is a plot of i) measured ionic current through a nanopore andii) measured nanowire FET conductance, respectively, as DNA translocatesthrough a nanopore in a nanopore sensor configured for local electricalpotential measurement, for a TMV of 0.6 V and 1:1 cis/trans reservoirsolution concentration ratio, with a local potential measurement made inthe trans reservoir; and

FIG. 25 is a plot of i) total ionic current measured through threenanopores sharing reservoirs, ii) measured nanowire FET conductancethrough the first of the nanopores, ii) measured nanowire FETconductance through the second of the nanopores, and ii) measurednanowire FET conductance through the third of the nanopores,respectively, as DNA translocates through the nanopores in the threesensors in a nanopore sensor configured for local electrical potentialmeasurement.

DETAILED DESCRIPTION

FIGS. 1A-1E are schematic views of nanopore sensor configurationsprovided herein that enable a local electrical potential sensing methodfor nanopore sensing. For clarity of discussion, device featuresillustrated in the figures are not shown to scale. Referring to FIG. 1A,there is shown a nanopore sensor 3 including a support structure 14,such as a membrane, in which is disposed a nanopore 12. The nanopore 12is configured in the support structure between two fluidic reservoirsshown here schematically as a trans reservoir and a cis reservoir suchthat the nanopore 12 is the only path of fluidic communication betweenthe cis and trans reservoirs. One reservoir is connected to an inlet tothe nanopore while the other reservoir is connected to an outlet fromthe nanopore. In operation of the nanopore sensor for local electricalpotential measurement detection of species translocation through thenanopore, one or more objects of a species, such as molecules, areprovided in a fluidic solution in one of the reservoirs fortranslocation through the nanopore to the other of the two reservoirs.For many applications, and in particular for molecular sensingapplications, it can be preferred to provide the molecules or otherspecies objects in an ionic fluidic solution in one of the reservoirs,and can be provided in either one of the reservoirs.

The species objects to be translocated through the nanopore can includeobjects selected from, for example, DNA, DNA fragments, RNA, RNAfragments, PNA, nucleotides, nucleosides, oligonucleotides, proteins,polypeptides, amino acids and polymers. The species objects can includea tag that is released from a tagged nucleotide. With the aid of apolymerase, nucleotides along a nucleic acid molecule can be polymerizedto generate a nucleic acid strand that is complementary to at least aportion of the nucleic acid molecule, whereby, during polymerization, atag is released from an individual nucleotide of the nucleotides, andwhereby the released tag translocates the nanopore, as described in WO2013/191793, hereby incorporated by reference.

The nanopore may be provided as an aperture, gap, channel, groove, poreor other hole in the support structure and is provided with an extent,such as a diameter, for a corresponding geometry, that is suitable forsensing species objects of interest. For sensing molecule translocationthrough the nanopore, a nanopore of less than about 100 nm can bepreferred, and a nanopore of less than 10 nm, 5 nm, or 2 nm can be morepreferred. As discussed below, a nanopore of 1 nm can be suitable andeven preferred for some molecular sensing applications.

The reservoirs or other components of the nanopore sensor may beconfigured to provide a driving force for moving objects of a species,such as molecules, toward the nanopore or through the nanopore from oneof the reservoirs to the other of the reservoirs. For example,electrodes 13, 15 can be provided in a circuit with voltage and currentelements 16, 18 to produce an electrophoretic force between thereservoirs for electrophoretically driving the species in the solution,towards the nanopore or through the nanopore from one reservoir to theother reservoir. To enable electrophoretic driving of the species, thefluidic solutions of the reservoirs can be provided as electricallyconductive ionic solutions having pH and other characteristics that areamenable to the species in the solution. Thereby an electrical circuitcan be connected with the reservoir solutions in series through thenanopore, with electrodes 13, 15 as shown in the figures, providing anelectrical voltage bias between the solutions, across the nanopore.Translocation and control of the rate of translocation of species thoughthe nanopore can be carried out with alternative techniques, such as anenzyme molecular motor.

In addition to or as an alternative to a driving force that is anapplied voltage, a pressure gradient across the pore can be used tobring molecules towards the nanopore and/or through the nanopore. Thispressure gradient can be produced by using a physical pressure, or achemical pressure such as an osmotic pressure. An osmotic pressure canbe produced from a concentration difference across the cis and transchambers. The osmotic pressure can be produced by having a concentrationgradient of an osmotically active agent, such as a salt, polyetheleneglycol (PEG), or glycerol.

As shown in FIG. 1A, there can be provided in the nanopore sensor atransduction element 7 that senses the electrical potential local to thesite of the element and that develops a characteristic that isindicative of that local electrical potential. An electrical connection,such as device or region of a device and/or circuit, a wire, orcombination of circuit elements, that senses the electrical potentiallocal to the site of the device and/or circuit can be provided as atransduction element 7, to develop a signal indicative of localelectrical potential. The location of the electrical potential sensingcan be in a reservoir, on a surface of the support structure, or otherlocation within the nanopore sensor as described in detail below.

As shown in FIG. 1B, there can be provided a circuit 20 that includes,e.g., a transistor device 22, having a source, S, a drain, D, and achannel region 24. The channel region 24 is in this example physicallydisposed at a location in the nanopore sensor environment to make alocal electrical potential measurement. This physical location of thechannel region 24 of the transistor can be at any convenient andsuitable site for accessing local electrical potential.

In the arrangements of FIGS. 1A-1B, an electrical potential sensingcircuit is configured local to the trans reservoir to provide atransistor or other device that measures the electrical potential localto the trans reservoir at the trans reservoir-side of the nanopore 12.Alternatively, as shown in FIG. 1C, an electrical transduction element7, such as an electrical potential sensing device or circuit, can beconfigured at the cis reservoir side of the nanopore. Here, e.g., asshown in FIG. 1D, there can be provided a circuit 20 including atransistor 24 or other device for measuring electrical potential localto the cis reservoir at the cis reservoir side of the nanopore 12.

In a further alternative configuration, as shown in FIG. 1E, there canbe included two or more transduction elements, with circuits 20 a, 20 b,etc., connected to transduction elements such as transistors 22 a, 22 bthat sense the electrical potential at two or more locations in thenanopore sensor system, such as each side of the nanopore supportstructure. Depending on the physical implementation of the electricalpotential sensing circuit, the electrical potential at the two sides ofthe nanopore membrane 14 can thereby be measured with this arrangement.This is an example configuration in which is enabled a measurement ofthe difference in local potential between two sites in the nanoporesensor. It is therefore intended that the term “measured localelectrical potential” refers to the potential at a single site in thenanopore sensor, refers to a difference or sum in local electricalpotential between two or more sites, and refers to a local potential attwo or more sites in the nanopore sensor configuration.

The local electrical potential measurement can be made by any suitabledevice and/or circuit or other transduction element, includingbiological or other non-solid state transduction elements, and is notlimited to the transistor implementation described above. As shown inFIG. 1F, there can be provided a transduction element on the supportstructure 14 that is configured as a single electron transistor (SET)circuit 27. The source, S, and drain, D, regions of the SET are disposedon the support structure, providing tunneling barriers to the SET 27. Inthe resulting quantum dot system, the electrical conductance through theSET 27 depends on the energy level of the SET with respect to the Fermilevel of the source, S, and drain, D. With the nanopore 12 located inthe vicinity of the SET, the electrical potential, and correspondingenergy level, of the SET changes as species objects translocate throughthe nanopore, changing the conductance of the SET circuit.

Further, as shown in FIG. 1G, there can be provided on the supportstructure 14 a quantum point contact (QPC) system 29 for making a localelectrical potential measurement. In this system, an electricallyconductive region 31 is provided that forms source, S, and drain, D,regions that are connected via a very thin conducting channel region atthe site of the nanopore 12. The channel region is sufficiently thinthat the electronic carrier particle energy states that areperpendicular to the channel region are quantized. As species objectstranslocate through the nanopore, the local potential around the QPC,thus the Fermi level inside the thin conduction channel region changes,resulting in a change in the number of quantized states below the Fermilevel, and a corresponding change in QPC conductance.

A nanowire FET can also be configured at the site of the nanopore. Thenanowire can be formed of any suitable electrically conducting orsemiconducting material, including fullerene structures andsemiconducting wires. The term “nanowire” as used herein refers to anelectrical conduction channel that is characterized by a width that iscompatible with the signal decay length measured from the nanopore site.The channel width is preferably on the same order of magnitude as thedecay length and can be larger. The nanowire can be made from anysemiconductor material that is stable in the selected reservoirsolution.

The nanopore sensor is not limited to solid state nanoporeconfigurations with solid state voltage sensing devices. Biologicalnanopores and potential sensing arrangements can also be employed, e.g.,with a protein nanopore or other suitable configuration. As shown inFIG. 1H, there can be provided an amphiphilic layer 31 in which isdisposed a protein nanopore 33. A voltage-sensitive dye, e.g., afluorescent direct dye 37, can be provided in the lipid bilayer as anelectrical transduction element. With this arrangement, when a speciesobject such as a molecule translocates through the protein nanopore, thevoltage drop across the amphiphilic layer changes, and the fluorescenceof the dye is modulated by the voltage change. Optical detection orsensing of the dye fluorescence and changes to that fluorescence providesensing of the electrical potential at the nanopore. Optical microscopyor other conventional arrangement can be employed for making thispotential measurement as an optical output signal from the nanoporesensor. This amphiphilic layer nanopore sensor is an example of abiological nanopore sensor that is based on sensing of the localpotential at a site in the nanopore system. The method of localpotential measurement for nanopore translocation detection is notlimited to a particular solid state or biological configuration and canbe applied to any suitable nanopore configuration.

The support structure can be formed from either or both organic andinorganic materials, including, but not limited to, microelectronicmaterials, whether electrically conducting, electrically semiconducting,or electrically insulating, including materials such as II-IV and III-Vmaterials, oxides and nitrides, like Si₃N₄, Al₂O₃, and SiO, organic andinorganic polymers such as polyamide, plastics such as Teflon®, orelastomers such as two-component addition-cure silicone rubber, andglasses. A solid state support structure may be formed from monatomiclayers, such as graphene, or layers that are only a few atoms thick suchas those disclosed in U.S. Pat. No. 8,698,481, and U.S. PatentApplication Publication 2014/174927, both hereby incorporated byreference. More than one support layer material can be included, such asmore than one graphene layer, as disclosed in US Patent ApplicationPublication 2013/309776, incorporated herein by reference. Suitablesilicon nitride membranes are disclosed in U.S. Pat. No. 6,627,067, andthe support structure may be chemically functionalized, such asdisclosed in U.S. patent application publication 2011/053284, bothhereby incorporated by reference.

For a selected support structure material composition, thickness, andarrangement, any suitable method can be employed for producing ananopore in the support structure. For example, electron beam milling,ion beam milling, material sculpting with an energetic beam, dryetching, wet chemical or electrochemical etching, or other method can beemployed for producing a nanopore, as described, e.g., in U.S. PatentApplication Publication 2014/0262820, U.S. Patent ApplicationPublication 2012/0234679, U.S. Pat. Nos. 8,470,408, 8,092,697,6,783,643, and 8,206,568, all of which are hereby incorporated byreference. In addition, extrusion, self-assembly, material deposition onsidewalls of a relatively large aperture, or other nanopore formationmethod can be employed.

As an alternative to providing a completely solid state nanopore, abiological nanopore can be provided within a solid state aperture. Sucha structure is disclosed for example in U.S. Pat. No. 8,828,211, herebyincorporated by reference. Further, a biological nanopore may be atransmembrane protein pore. The biological pore may be a naturallyoccurring pore or may be a mutant pore. Typical pores are described inU.S. Patent Application No. 2012/1007802, and are described in StoddartD et al., Proc Natl Acad Sci, 12;106(19):7702-7, Stoddart D et al.,Angew Chem Int Ed Engl. 2010;49(3):556-9, Stoddart D et al., Nano Lett.2010 Sep. 8;10(9):3633-7, Butler TZ et al., Proc Natl Acad Sci2008;105(52):20647-52, U.S. Patent Application Publication 2014/186823,and WO2013/153359, all of which are hereby incorporated by reference.The pore may be homo-oligomeric, namely, derived from identicalmonomers. The pore may be hetero-oligomeric, namely where at least onemonomer differs from the others. The pore may be a DNA origami pore, asdescribed by Langecker et al., Science, 2012; 338: 932-936, herebyincorporated by reference.

In one embodiment the pore can be provided within an amphiphilic layer.An amphiphilic layer is a layer formed from amphiphilic molecules, suchas phospholipids, which have both hydrophilic and lipophilic properties.The amphiphilic layer can be a monolayer or a bilayer, with the layerselected from a lipid bilayer or non-natural lipid bilayer. The bilayercan be synthetic, such as that disclosed by Kunitake T., Angew. Chem.Int. Ed. Engl. 31 (1992) 709-726. The amphiphilic layer can be aco-block polymer such as disclosed by Gonzalez-Perez et al., Langmuir,2009, 25, 10447-10450, and U.S. Pat. No. 6,723,814, both herebyincorporated by reference. The polymer can be, e.g., a PMOXA-PDMS-PMOXAtriblock copolymer.

Referring to FIG. 2A, any of these support structures, nanopores, andelectrical configurations for measuring the local electrical potentialat one or more sites in a nanopore sensor can be employed in a methodfor sensing the translocation of species through the nanopore. Toexplain the principle of this sensing, it is instructive to model thenanopore sensor as a circuit 35 including electrical componentscorresponding to physical elements of the sensor, as shown in FIG. 2A.The cis and trans reservoirs can each be modeled with a characteristicfluidic access resistance, R_(Trans), 36, R_(Cis), 38. This accessresistance is defined for this analysis as the fluidic resistance in areservoir solution local to the site of the nanopore, not in the bulksolution away from the nanopore. The nanopore can be modeled with acharacteristic nanopore solution resistance, R_(Pore), 40 that is thefluidic resistance of solution through the length of the nanoporebetween the two sides of the support structure in which the nanopore isdisposed. The nanopore can also be modeled with a characteristiccapacitance C_(Pore), that is a function of the membrane or othersupport structure in which the nanopore is disposed. The accessresistance of both chambers and the nanopore solution resistance arevariable.

In a nanopore sensor starting condition in which no species aretranslocating the nanopore, the nanopore can be characterized by thesolution resistance, R_(Pore), given above, and both fluidic reservoirscan be characterized by the access resistances of the trans reservoirand the cis reservoir, R_(Trans) and R_(Cis), respectively. Then when aspecies object, such as a biological molecule 45, translocates thenanopore 12 as shown in FIG. 2A, the solution resistance, R_(Pore), ofthe nanopore and the access resistances, R_(Trans) and R_(Cis), of eachof the reservoirs, change because the molecule in the nanopore at leastpartially blocks the passageway through the nanopore length, changingthe effective diameter of the nanopore. With such a blockage, thefluidic solution resistance of the nanopore and the access resistance ofboth reservoirs increase above the resistance of the nanopore and accessresistance of both reservoirs with no molecule present in the nanopore.

The partial blockage of the nanopore by a species object effects thenanopore solution resistance and the reservoir access resistancesdifferently, as explained in detail below. As a result, the partialblockage of the nanopore by a translocating species causes acorresponding redistribution of electrical voltage occurs between thenanopore and the cis and trans reservoirs solutions, and the electricalpotential at sites throughout the nanopore sensor accordingly adjusts.The local electrical potential at both the sites denoted as A and B inFIG. 2A thereby changes accordingly with this change in nanoporesolution resistance and redistribution of voltage between the reservoirsolutions and the nanopore. A measurement of electrical potential ateither of these sites, or at another site of the nanopore sensorconfiguration, or a measurement of a difference in local potentialbetween two or more sites, thereby provides an indication of thetranslocation of the molecule through the nanopore.

The local electrical potential at a selected nanopore sensor site andchanges in this potential can be sensed by an electrical transductionelement disposed in the nanopore sensor. For example, changes in theconductance of the conducting channel in a transistor device can providean electrical potential measurement. Transistor channel conductancetherefore can be employed as a direct indication of the electricalpotential local to the physical location of the transistor channel. Thenanopore sensor arrangements of FIGS. 1A-1B correspond to a localelectrical potential measurement at site A in the circuit 35 of FIG. 2A.The nanopore sensor arrangements of FIG. 1C-1D correspond to a localelectrical potential measurement at site B in the circuit 35 of FIG. 2A.The nanopore sensor arrangement of FIG. 1E corresponds to a localelectrical potential measurement at both sites A and B in the circuit 35of FIG. 2A, and enables a determination of the difference between thepotential at those two sites.

An electrical circuit equivalent of the example configuration of FIG. 1Bis shown in FIG. 2B. Here is represented the access resistances of thecis and trans reservoirs, R_(Cis), R_(Trans), respectively, and thefluidic solution resistance, R_(Pore), of the nanopore. The location ofan electrical transduction element for measuring local potential, e.g.,the channel of a transistor 22, is here positioned at the site A in FIG.2A, providing a local electrical potential indication in the transreservoir at the trans reservoir side of the nanopore. With thisarrangement, as species objects such as molecules translocate throughthe nanopore, the output signal of the electrical potential measurementcircuit can be monitored for changes in electrical potential,corresponding to changes in the state of the nanopore and the presenceor absence of one or more objects in the nanopore.

This analysis can be applied to any nanopore sensor in which there isprovided a local electrical transduction element. The analysis is notlimited to the FET and other implementations described above, and isapplicable to any suitable arrangement for any transduction element. Allthat is required is the provision of an electrical transduction element,such as a device, region of a device, circuit, or other transductionelement that makes a local electrical potential measurement as speciesobjects translocate the nanopore.

To further analyze the nanopore sensor system parameters, the nanoporesensor can be modeled as shown in the schematic representation of FIG.3A. Several assumptions can be employed to enable an analyticalcalculation. First, geometrical changes of the nanopore supportstructure, such as a membrane, as well as the nanopore itself, and otherregions of the nanopore sensor, caused by the inclusion of a localpotential sensing transduction element can be ignored and the potentialsensing transduction element can be modeled as a point potentialdetector. The fluidic reservoirs are assumed to include electricallyconductive ionic solutions. The two reservoir solutions are specified toinclude distinct ionic concentrations that may be differing ionicconcentrations. In one analysis, with differing ionic concentrationsspecified for the reservoirs, the ionic concentration distributionthrough the nanopore system is determined by the steady state diffusionthat is driven by the cis/trans reservoir concentration difference; thediffusion is assumed to reach steady state. A further assumption can bemade by approximating the buffer concentration distribution andelectrical potential as being constant in small hemispheres on bothsides of the nanopore. The nanopore sensor is assumed to be in steadystate. Under these conditions, the diffusion equations of the nanoporesensor are given as:

                                           (1) $\left\{ \begin{matrix}{\frac{\partial C}{\partial t} = 0} & \left( {{For}\mspace{14mu} {both}\mspace{14mu} {chambers}\mspace{14mu} {and}\mspace{14mu} {inside}\mspace{14mu} {the}\mspace{14mu} {nanopore}} \right) \\{{{r\frac{\partial^{2}{C(r)}}{\partial r^{2}}} + {2\frac{\partial{C(r)}}{\partial r}}} = 0} & {\ \left( {{For}\mspace{20mu} {both}{\mspace{11mu} \ }{chambers}} \right)\ } \\{\frac{\partial{C(z)}}{\partial z} = \ {const}} & {\ \left( {{Inside}\mspace{14mu} {the}\mspace{14mu} {nanopore}} \right)}\end{matrix} \right.$

Where C is fluidic ion concentration, t is time, r is location in areservoir at a point measured from the nanopore, and z is distancethrough the nanopore length. If these diffusion equations are solvedunder the boundary conditions that in the cis reservoir far away fromthe nanopore, C=C_(cis), in the trans reservoir far away from thenanopore, C=C_(Trans), the flux is the same in the nanopore and for bothreservoirs, and the concentration is continuous at the nanopore openingin each reservoir, then the ionic concentration of the two reservoirsand the nanopore can be given as:

$\begin{matrix}{\quad\left\{ \begin{matrix}{{C_{C}(r)} = {C_{Cis} - {\frac{C_{Cis} - C_{Trans}}{4\left( {{2l} + d} \right)}\frac{d^{2}}{r}}}} & \left( {{Cis}\mspace{14mu} {chamber}} \right) \\{{C_{T}(r)} = {C_{Trans} - {\frac{C_{Trans} - C_{Cis}}{4\left( {{2l} + d} \right)}\frac{d^{2}}{r}}}} & \left( {{Trans}\mspace{14mu} {chamber}} \right) \\{{C_{P}(z)} = {C_{Trans} + {\frac{C_{Cis} - C_{Trans}}{2\left( {{2l} + d} \right)}\left( {{4\; z} + d} \right)}}} & ({Nanopore})\end{matrix} \right.} & (2)\end{matrix}$

Here l and d are thickness of the nanopore support structure andnanopore diameter, respectively. Because the ionic concentrationdistribution is therefore known and the solution conductivity isproportional to the concentration, then the conductivity of solution, σ,is given as:

σ=Σ·C.  (3)

Here Z is the molar conductivity of solution. Assuming the total currentis I, then the electrical potential drop through the nanopore sensor,with a cis reservoir voltage, V_(C), a trans reservoir voltage, V_(T),and a nanopore voltage, V_(P), can be given as:

$\begin{matrix}{\quad\left\{ \begin{matrix}{{d{V_{C}(r)}} = \frac{Idr}{2\; \pi \; \Sigma \; {C_{C}(r)}r^{2}}} & \left( {{Cis}\mspace{14mu} {chamber}} \right) \\{{d{V_{T}(r)}} = \frac{- {Idr}}{2{\pi\Sigma}\; {C_{T}(r)}r^{2}}} & \left( {{Trans}\mspace{14mu} {chamber}} \right) \\{{d{V_{P}(r)}} = \frac{4{Idz}}{{\pi\Sigma}\; {C_{P}(z)}d^{2}}} & ({Nanopore})\end{matrix} \right.} & (4)\end{matrix}$

If these three equations are solved with boundary conditions specifyingthat far away from nanopore in the cis reservoir the electricalpotential equals the voltage applied across the structure or membrane,i.e., a transmembrane voltage (TMV), to electrophoretically drive anobject through the nanopore, and that far away from nanopore in thetrans chamber, the potential is 0 V, then the voltages in the nanoporesensor, namely, the voltage in the cis reservoir, V_(C)(r), the voltagein the trans reservoir, V_(T)(r), and the voltage in the nanopore,V_(P)(r), are given as:

$\begin{matrix}\left\{ \begin{matrix}{{V_{C}(r)} = {V + {\frac{2\; {I\left( {{2l} + d} \right)}}{\pi \; {\Sigma \left( {C_{Cis} - C_{Trans}} \right)}d^{2}}{\ln \left( {1 - \frac{\left( {C_{Cis} - C_{Trans}} \right)d^{2}}{4\left( {{2l} + d} \right)C_{Cis}r}} \right)}}}} \\{{V_{T}(r)} = {\frac{2\; {I\left( {{2l} + d} \right)}}{\pi \; {\Sigma \left( {C_{Cis} - C_{Trans}} \right)}d^{2}}{\ln \left( {1 + \frac{\left( {C_{Cis} - C_{Trans}} \right)d^{2}}{4\left( {{2l} + d} \right)C_{Trans}r}} \right)}}} \\{{V_{P}(r)} = {\frac{2\; {I\left( {{2l} + d} \right)}}{\pi \; {\Sigma \left( {C_{Cis} - C_{Trans}} \right)}d^{2}}{\ln\left( \frac{\begin{matrix}{{\left( {{4\; l} + d} \right)C_{Trans}} + {d\; C_{Cis}} +} \\{4\left( {C_{Cis} - C_{Trans}} \right)z}\end{matrix}}{2\left( {{2l} + d} \right)C_{Trans}} \right)}}}\end{matrix} \right. & (5)\end{matrix}$

Because the electrical potential is continuous at both nanopore openingsinto the reservoirs and because the total voltage applied is V,Expression (5) can be further simplified to:

$\begin{matrix}\left\{ \begin{matrix}{{V_{C}(r)} = {V + {\frac{V}{\ln \left( {C_{Cis}\text{/}C_{Trans}} \right)}{\ln \left( {1 - \frac{d^{2}\left( {1 - {C_{Trans}\text{/}C_{Cis}}} \right)}{4\left( {{2l} + d} \right)r}} \right)}}}} \\{{V_{T}(r)} = {\frac{V}{\ln \left( {C_{Cis}\text{/}C_{Trans}} \right)}{\ln \left( {1 + \frac{d^{2}\left( {{C_{Cis}\text{/}C_{Trans}} - 1} \right)}{4\left( {{2l} + d} \right)r}} \right)}}} \\{{V_{P}(r)} = {\frac{V}{\ln \left( {C_{Cis}\text{/}C_{Trans}} \right)}{\ln\left( \frac{\begin{matrix}{{4\; l} + d + {d\; C_{Cis}\text{/}C_{Trans}} +} \\{4\left( {{C_{Cis}\text{/}C_{Trans}} - 1} \right)z}\end{matrix}}{2\left( {{2l} + d} \right)} \right)}}}\end{matrix} \right. & (6)\end{matrix}$

With this expression, the electric field, E_(P)(r) inside nanopore canbe given as:

$\begin{matrix}{{{E_{P}(r)} = {\frac{d{V_{P}(r)}}{dz} = \frac{4{V\left( {{C_{Cis}\text{/}C_{Trans}} - 1} \right)}}{\ln \left( {C_{Cis}\text{/}C_{Trans}} \right)}}}{\frac{1}{\begin{matrix}{{4l} + d + {{dC}_{Cis}\text{/}C_{Trans}} +} \\{4\left( {{C_{Cis}\text{/}C_{Trans}} - 1} \right)z}\end{matrix}}.}} & (7)\end{matrix}$

With this expression, the electrical potential change at the transreservoir side of the nanopore can be estimated by the electricalpotential change due to a reduction in the nanopore area, A, by thepresence of a species object, such as a molecule, in the nanopore, as:

$\begin{matrix}{\left. {\delta V_{T}} \right|_{d/2} = {{\frac{\partial V_{T}}{\partial A}{_{d/2}{{\delta \; A} = {\frac{2\delta \; A\; V}{{\pi ln}\left( {C_{Cis}\text{/}C_{Trans}} \right)}\frac{\left( {{4l} + d} \right)\left( {{C_{Cis}\text{/}C_{Trans}} - 1} \right)}{\begin{matrix}\left( {{2l} + d} \right) \\\begin{pmatrix}{{d^{2}\left( {{C_{Cis}\text{/}C_{Trans}} - 1} \right)} +} \\{4\left( {{2l} + d} \right)r}\end{pmatrix}\end{matrix}}}}}_{d/2}} = {\frac{2\delta \; A\; V}{{\pi ln}\left( {C_{Cis}\text{/}C_{Trans}} \right)}\frac{\left( {{4l} + d} \right)\left( {{C_{Cis}/C_{Trans}} - 1} \right)}{\left( {{2l} + d} \right)\left( {{d^{2}\left( {{C_{Cis}\text{/}C_{Trans}} + 1} \right)} + {4ld}} \right)}}}} & (8)\end{matrix}$

Here δA is the cross-sectional area of the molecule. The resistances ofthe nanopore sensor, namely, R_(Cis), R_(Trans), and R_(Pore), can becomputed based on the above expressions for voltage drop across thereservoirs and the nanopore as:

$\begin{matrix}\left\{ \begin{matrix}{R_{Cis} = {\frac{{- 2}\left( {{2l} + d} \right)}{\pi \; {\Sigma \left( {C_{Cis} - C_{Trans}} \right)}d^{2}}\ln \; \left( {1 - \frac{\left( {C_{Cis} - C_{Trans}} \right)d}{2\left( {{2l} + d} \right)C_{Cis}}} \right)}} \\{R_{Trans} = {\frac{2\left( {{2l} + d} \right)}{\pi \; {\Sigma \left( {C_{Cis} - C_{Trans}} \right)}d^{2}}\ln \; \left( {1 + \frac{\left( {C_{Cis} - C_{Trans}} \right)d}{2\left( {{2l} + d} \right)C_{Trans}}} \right)}} \\{R_{Pore} = {\frac{2\left( {{2l} + d} \right)}{\pi \; {\Sigma \left( {C_{Cis} - C_{Trans}} \right)}d^{2}}\ln \; \left( \frac{{\left( {{4l} + d} \right)C_{Cis}} + {dC_{Trans}}}{{\left( {{4l} + d} \right)C_{Trans}} + {dC_{Cis}}} \right)}}\end{matrix} \right. & (9)\end{matrix}$

So, the total resistance and ionic current of the nanopore sensor aregiven as:

$\begin{matrix}\left\{ {\begin{matrix}{R = {{R_{Cis} + R_{Trans} + R_{Pore}} = {\frac{2\left( {{2l} + d} \right)}{\pi \; {\sum{\left( {C_{Cis} - C_{Trans}} \right)d^{2}}}}{\ln \left( \frac{C_{Cis}}{C_{Trans}} \right)}}}} \\{I = {{V/R} = \frac{\pi \; {\sum{\left( {C_{Cis} - C_{Trans}} \right)d^{2}V}}}{2\left( {{2l} + d} \right){\ln \left( {C_{Cis}/C_{Trans}} \right)}}}}\end{matrix}.} \right. & (10)\end{matrix}$

With these expressions, it is demonstrated that the electricalcharacteristics of the nanopore sensor, and in particular thedistribution of electrical potential in the sensor, depends directly onthe ionic concentration of the fluidic solutions in the cis and transreservoirs. Specifically, the ratio of the reservoir solutionconcentrations directly impacts the magnitude of the change in localpotential due to species translocation of the nanopore.

FIGS. 3B-3E are plots of electrical potential and electric field in thenanopore, demonstrating these conditions. Given a cis/trans buffersolution concentration ratio=1:1, a 50 nm-thick nitride membrane, a 10nm diameter nanopore in the membrane, and a 1 V transmembrane voltage,i.e., 1 V applied between the solutions in the two reservoirs, then theelectrical potential in the nanopore as a function of distance from thenanopore opening at the cis reservoir is plotted in FIG. 3B, based onExpression (6) above. That same potential is plotted in FIG. 3C for acondition in which the cis/trans buffer solution concentration ratio isinstead 100:1. Note the increase in electrical potential at a givennanopore location for the unbalanced buffer solution ratio at pointscloser to the lower-concentration reservoir.

FIG. 3D is a plot of the electric field in the nanopore under theconditions given above, here for a balanced buffer solution ratio, basedon Expression (7) above. That same electric field profile is plotted inFIG. 3E for a condition in which the cis/trans buffer solutionconcentration ratio is instead 100:1. Note the increase in electricalpotential at a given nanopore location for the unbalanced buffersolution ratio, and that the electric field is dramatically stronger atpoints closer to the low concentration, higher resistance, reservoir.

With this finding, it is discovered that with a condition in which thereservoir solutions are both provided as electrically-conductive ionicsolutions of the same ionic concentration, the ratio of the accessresistance of the cis reservoir, R_(Cis), the access resistance of thetrans reservoir, R_(Trans), and the solution resistance of the nanopore,R_(Pore), are all fixed and the nanopore resistance is much larger thanthe reservoir access resistances. But under non-balanced ionconcentration conditions, the reservoir having a lower ionicconcentration has a larger access resistance, that can be on the orderof the nanopore resistance, while the higher-ionic concentrationreservoir resistance becomes comparably negligible.

Based on a recognition of this correspondence, it is herein discoveredthat to maximize a local potential measurement in the nanopore sensor,in one embodiment, the ionic reservoir solutions can be provided withdiffering ion concentrations. With this configuration of unbalancedionic concentration, the local potential measurement is preferably madeat a site in the reservoir which includes the lower ionic concentration.It is further preferred that the buffer concentration of the lower-ionconcentration solution be selected to render the access resistance ofthat reservoir of the same order of magnitude as the nanopore resistanceand much larger than the resistance of the high-ion concentrationsolution, e.g., at least an order of magnitude greater than that of thehigh-ion concentration solution, so that, for example, if the localpotential measurement is being made in the trans reservoir:

R_(T), R_(P)>>R_(C)  (11)

Based on this discovery then, for a given nanopore diameter, which setsthe nanopore resistance, R_(P), it is preferred in one embodiment todecrease the ionic solution buffer concentration of the reservoirdesignated for local potential measurement to a level at which theaccess resistance of that reservoir is of the same order of magnitude asthe nanopore resistance. This reservoir access resistance should notdominate the nanopore sensor resistance, but should be on the order ofthe nanopore resistance.

This condition can be quantitatively determined directly by electricallymodelling the nanopore sensor components in the manner described above.Based on Expression (8) above, there can be determined the ratio ofsolution concentrations that maximize the potential change duringnanopore translocation of a selected object for given nanopore sensorparameters. FIG. 4A is a plot of Expression (8) for a 50 nm-thicknanopore membrane and a configuration of a 1 V TMV for electrophoreticspecies translocation as a dsDNA molecule translocates through thenanopore. The potential change is shown as a function of theC_(Cis)/C_(Trans) ionic concentration ratio for various nanoporediameters below 10 nm. From this plot, it is found that the localpotential change in the trans reservoir is maximized for a ˜100:1C_(Cis)/C_(Trans) chamber buffer concentration ratio for any nanoporediameter modelled here. FIG. 4B is a plot of the correspondingcalculated potential change distribution in the trans reservoir for a 10nm-diameter nanopore at 1 V TMV for the selected 100:1 C_(Cis)/C_(Trans)solution concentration ratio.

This demonstrates that based on the discovery herein, in one embodiment,for a given reservoir site that is selected for making electricalpotential measurements, the ratio of ionic fluid buffer concentration inthe two reservoirs can be selected with the lower buffer concentrationsolution in the measurement reservoir, to maximize the amplitude of theelectrical potential changes at that selected measurement site. Thedistribution of this resulting potential change is highly localizedwithin several tens of nanometers of the nanopore, as shown in FIG. 4B.For the 100:1 C_(Cis)/C_(Trans) solution concentration ratio andnanopore parameters given just above, it can be determined, e.g., basedon Expression (9) above, that the access solution resistance of thetrans reservoir and the solution resistance of the nanopore are indeedwithin the same order of magnitude.

With this arrangement of reservoir fluidic solution bufferconcentrations and potential measurement configuration, it is noted thatthe local potential sensing technique produces a local potentialmeasurement signal that depends on the trans-membrane voltage (TMV) andthe ionic current signal. Other sensor based nanopore technologiesgenerally rely on a direct interaction between a translocating speciesand the nanopore sensor through, e.g., electrical coupling or quantummechanical tunnelling. For these techniques, the nanopore output signalis typically not directly related to the TMV or ionic current and shouldnot change significantly when the TMV is changed. In contrast, in thelocal potential measurement system herein, the nanopore sensor signal isproportional to the TMV and can be regarded as a linear amplification ofthe ionic current signal. As a result, both the local potentialmeasurement signal and the ionic current signal amplitudes depend on theTMV linearly, but the ratio between them is a constant for a givennanopore geometry and reservoir solution concentrations, as evidenced bythe expressions given above.

An advantage of the local potential measurement method is thecharacteristically high-bandwidth capability of the measurement with lownoise. Low signal bandwidth is one of the issues that limits directnanopore sensing by the conventional ionic current blockage measurementtechnique, due to the difficulties of high bandwidth amplification ofvery small measured electrical current signals. This can be particularlytrue for a small nanopore when employed for DNA sensing. In the localpotential sensing method, a large local electrical potential signal ismeasured instead of a small current signal, so the signal bandwidth isnot limited by the capabilities of a current amplifier. As a result,high-bandwidth signal processing electronics can be integrated on asolid state nanopore sensing structure.

Further, except for intrinsic shot noise and Johnson noise, the majorityof noise contributions to an ionic current blockage measurementtechnique are introduced through the capacitive coupling cross ananopore membrane and this capacitive coupling component of noise can beoverwhelming at certain stages of nanopore operation. Conventionally, avery small membrane area exposure to a reservoir solution is required inan effort to minimize noise. In the local potential measurement methodherein, because the local potential signal decays within a few tens ofnanometers around the nanopore for reasonable reservoir concentrationratios, the local potential measurement signal is only affected bycapacitive coupling between reservoir solutions within this localvolume. Therefore, the majority of capacitive coupling noise isautomatically rejected in the local electrical potential measurementsensing method.

Referring to FIGS. 4C-4D, the reservoir buffer solution concentrationratio can be selected, in one embodiment, to optimize the signalbandwidth of the nanopore sensor. Given that the local potentialmeasurement is to be made on one side of the nanopore, say the transside of the nanopore, then the cis reservoir solution concentration isset as high as reasonable, e.g., about 4 M, about a saturated solution,to minimize the nanopore solution resistance. Then, the signal noise asa function of bandwidth is analyzed, e.g., based on the plot of FIG. 4C.Here are plotted the various contributions to noise as well as thesignal expected for a fluidic nanopore operation. The plot labelled“free space” refers to a computation based on free-space molecular size.The plot labelled “Bayley” refers to computation based on molecular sizefrom previous work by Bayley et al, in J. Clarke et al., “ContinuousBase Identification for Single-Molecule Nanopore DNA Sequencing,” NatureNanotechnology, N. 4, pp. 265-270, 2009. The two signal lines are theminimum signal difference that is attained between the four DNA bases,which minimum signal exists between the A and the T bases. Here thenanopore is given as a 1 nm-diameter nanopore in a graphene membrane,with a 4 M cis reservoir solution concentration, a buffer concentrationratio between the reservoirs of 50:1, and a voltage noise density ofabout 10⁻⁹ V/√Hz. The dielectric loss factor for graphene is unknown, so1 was used for convenience. Finding the cross point of the signal andtotal noise in the plot sets the 1:1 signal-to-noise ratio (S/N). Thisis the highest possible signal bandwidth. For example, for the fluidicnanopore operation, the 1:1 S/N ratio is at a bandwidth of about 100MHz. A bandwidth greater than about 50 MHz can be preferred as well asthe 100 MHz bandwidth.

Referring to the plot of FIG. 4D, the 100 MHz bandwidth corresponds toreservoir solution concentration ratio of about 50:1, where the localpotential is to be made in the low-concentration reservoir side of thenanopore. For the nanopore sensor parameters used in this example, anyreservoir concentration ratio higher than about 50:1 will decrease thebandwidth. Any concentration ratio lower than about 50:1 will decreasethe signal-to-noise ratio. Therefore, it is discovered herein that thebandwidth can be optimized and there exists an optimization point ofreservoir concentration ratio, say 50:1. The reservoir solutionconcentration ratio therefore can be selected, in one embodiment, basedon a trade-off between the characteristic noise of the nanopore sensorand the desired operational bandwidth of the nanopore sensor. It is tobe recognized therefore that to minimize noise, the reservoir solutionconcentration ratio can be increased, but that the bandwidth may becorrespondingly reduced. Alternatively, electronic signal processing,such as low-pass filtering, or other processing of the signal, can beemployed.

It is further to be recognized that in general, a nanopore of smallerdiameter produces a larger signal for a given species object totranslocate through the nanopore. For applications such as sensing aparticular molecule, like DNA, however, the nanopore extent ispreferably based on the molecule extent, and the tuning of the reservoirconcentration ratio is made accordingly.

The reservoir buffer solution concentration ratio can also be selected,in a further embodiment, to produce a signal decay length, measured fromthe site of the nanopore, that accommodates a selected local potentialmeasurement device. It is recognized that the decay length of the signalshould be sufficiently large to accommodate the arrangement of apotential measurement device within the decay length. FIG. 4E is a plotof signal decay length for a range of buffer concentration ratios, giventhat the local potential measurement is to be made on the transreservoir side of the nanopore. The plot is based on the circuit modelshown inset in the plot.

Based on this analysis, it is shown that at concentration ratios greaterthan about 20 or 30, there is produced a sufficient signal decay lengthto accommodate a device that can measure the local electrical potentialwithin the decay length. At concentration ratios greater than about50:1, ample decay length is provided for making a potential measurementwithin the decay length. A signal decay length greater than about 5 nmcan be preferred, as well as a signal decay length of, e.g., about 10 nmand about 20 nm.

This particular embodiment with a nanopore sensor analysis forspecifying the relative cis and trans solution ionic concentrations isbased on a consideration of the changes in nanopore resistance andreservoir access resistances that are caused by species translocationthrough a nanopore. The methodology herein provides a further analysis,methods, and structures that additionally consider changes in theresistance of the cis and trans reservoir fluids away from the immediatevicinity of the nanopore during species translocation.

Referring to FIG. 5, in this further embodiment, there is schematicallyshown a nanopore sensor 100 provided herein, the nanopore sensor 100including a cis reservoir 102 and a trans reservoir 104 on opposingsides of a nanopore 12 that is disposed in a support structure 14. Thenanopore must be traversed in a path of fluidic communication betweenthe two reservoirs. A fluidic passage 105 is disposed between onereservoir, here shown as the trans reservoir, and the nanopore 12 tofluidically connect that reservoir to the nanopore through the fluidicpassage. The fluidic passage has a passage length that is greater thanpassage cross-sectional extent, width, or diameter, and is connected toenable fluidic communication between the nanopore and the reservoir towhich the passage leads. The second reservoir, here the cis reservoir,is arranged for fluidic communication with the fluidic passage by way ofthe nanopore. The second reservoir does not need to include a secondfluidic passage.

As in the nanopore sensor examples described above, the nanopore sensor100 of this embodiment can include electrodes 13, 15, and a voltagesource 16 for applying an electrical potential between the reservoirs,across the solid state support structure. The nanopore is disposed inthe suitable support structure and is solid state, biological, or somecombination of the two in the manner described above.

This nanopore sensor embodiment can be electrically modelled as shown inthe circuit of FIG. 6. In this model, the aspect ratio of the nanopore,i.e., the ratio of nanopore diameter and length, and the aspect ratio ofthe fluidic passage are a priori specified to be sufficiently large thatthe fluidic access resistance of the trans and cis reservoir chamberscan be ignored. There is then defined a resistance of the fluidicpassage, R_(FP), and the nanopore resistance, R_(PORE). It is further apriori specified that the fluidic resistance of the fluidic passage,R_(FP), is much larger than the bulk fluidic resistance of the cisreservoir and is much larger than the bulk fluidic resistance of thetrans reservoir. As a result, as shown in FIG. 6 there can be modelledthe nanopore sensor resistance as a fluidic passage resistance and ananopore resistance.

Referring to both FIG. 6 and FIG. 5, an electrical transduction element7, like that described above, is provided in the sensor to sense theelectrical potential local to fluidic passage 105, to develop acharacteristic that is indicative of the local electrical potential inthe fluidic passage. An electrical connection, such as device or regionof a device and/or circuit, a wire, or combination of circuit elements,that senses the electrical potential local to the site of the deviceand/or circuit can be provided as a transduction element to develop asignal indicative of local electrical potential. The location of theelectrical transduction element 7 can be in a reservoir, on a surface ofthe support structure, as shown in FIG. 5, at a location within thefluidic passage, or other location within the nanopore sensor.

As shown in FIG. 6, in one embodiment, there can be provided a circuit20 for supporting an electrical transduction element that is, e.g., atransistor device, having a source, S, a drain, D, and a channel region.The channel region can be physically disposed at a location in thenanopore sensor environment to make a local electrical potentialmeasurement. This physical location of the channel region of thetransistor can be at any convenient and suitable site for accessinglocal electrical potential.

Now considering this nanopore sensor arrangement, if the resistance ofthe fluidic passage 105 is comparable to the resistance of the nanopore12, then local electrical potential measurement of the fluidic passagemaximizes signal measurements indicative of species translocationthrough the nanopore in the manner discussed above, with reference tothe access resistance of one reservoir relative to the nanopore, as inExpression (11). With a measurement of local potential at the site shownin the circuit of FIGS. 5-6, then the sensed voltage, V_(Sens), is givenas:

V _(Sens) =V ₀ R _(FP)/(R _(Pore) +R _(FP))  (12)

where R_(Pore) is the resistance of the nanopore, R_(FP) is theresistance of the fluidic passage, and V₀ is the voltage 16 appliedacross the nanopore from the circuit.

The voltage signal, V_(Sig), to be detected during species translocationthrough the nanopore is the small voltage change that is caused by thecorrespondingly small resistance change of the nanopore due to thepartial blockage of the nanopore by the translocating species, withV_(Sig) given as:

$\begin{matrix}{V_{Sig} = {\frac{\partial V_{Sens}}{\partial R_{Pore}}d{R_{Pore}.}}} & (13)\end{matrix}$

This signal is maximized when

${\frac{\partial V_{Sig}}{\partial R_{FP}} = 0},$

requiring R_(FP)=R_(Pore). This relation demonstrates that if thefluidic passage resistance is not the same as the nanopore resistance,then the voltage signal that can be detected is smaller than a possiblemaximized voltage signal. The ratio, R_(Signal), between the actualvoltage signal that is attained and the maximized measured voltagesignal tells how far the system is away from the optimal condition, withthe ratio metric given as:

$\begin{matrix}{R_{Signal} = \frac{4\left( {R_{FP}/R_{Pore}} \right)}{\left( {1 + \frac{R_{FP}}{R_{Pore}}} \right)^{2}}} & (14)\end{matrix}$

Expression (14) is plotted in FIG. 7. This plot indicates that when thefluidic passage resistance is 10% of the nanopore resistance, thevoltage signal, V_(Sig), is more than 30% of the maximum attainablesignal. Thus, for purposes of this analysis, a condition in which whenthe fluidic passage resistance, R_(FP), is at least about 10% of thenanopore resistance, R_(Pore), and is no more than about 10 times thenanopore resistance, is considered to be matching of the fluidic passageresistance and the nanopore resistance.

To determine the structural and geometric requirements of the fluidicpassage that meet these resistance matching requirements, it can in oneexample methodology be assumed that both the nanopore and the fluidicpassage are generally cylindrical in geometry. Further, as stated above,the access resistance of the cis and trans reservoirs can be ignored forthis analysis, given that the aspect ratio of the nanopore and thefluidic passage are relatively high relative to the cis and transreservoirs. Finally, it is assumed that diffusion of ionicconcentrations between the cis and trans reservoirs is in a steady statecondition if the ionic concentrations in the two reservoirs aredifferent.

FIG. 8 is a schematic view defining the geometry of the fluidic passage105. Here l₁, r₁ and l₂, r₂ are the length and the radius of thenanopore and the fluidic passage, respectively. C_(C), C_(I) and C_(T)are the ionic solution concentrations in the cis chamber, the ionicsolution concentration at the interface between the nanopore and thefluidic passage, and the ionic concentration in the trans chamber,respectively.

Under steady state conditions, the diffusion flux, Flux, of ionicconcentration in the nanopore and the fluidic passage is equal, in acondition stated as:

$\begin{matrix}{{{Flux} \propto {\frac{C_{C} - C_{I}}{l_{1}}r_{1}^{2}}} = {{\frac{C_{I} - C_{T}}{l_{2}}r_{2}^{2}} = {const}}} & (15)\end{matrix}$

whereby the interface concentration, C_(I), can be determined as:

$\begin{matrix}{C_{I} = \frac{{C_{C}l_{2}r_{1}^{2}} + {C_{T}l_{1}r_{2}^{2}}}{{l_{2}r_{1}^{2}} + {l_{1}r_{2}^{2}}}} & (16)\end{matrix}$

so that the ionic concentration in the nanopore, C_(Pore), and the ionicconcentration in the fluidic passage, C_(FP), are given as:

$\begin{matrix}\left\{ \begin{matrix}{C_{Pore} = {{\frac{C_{C} - C_{I}}{l_{1}}x_{1}} + C_{I}}} \\{C_{D} = {C_{I}\  - {\frac{C_{I} - C_{T}}{l_{2}}x_{2}}}}\end{matrix} \right. & (17)\end{matrix}$

Under normal measurement conditions, the conductivity, σ, of a solutionis in general approximately proportional to the ionic concentration ofthe solution, as:

σ=Σ·C,

where Σ is the molar conductivity of the solution and C is the ionicconcentration. With this relation, the resistance, R_(Pore), of thenanopore and the resistance, R_(FP), of the fluidic passage, are givenas:

$\begin{matrix}\left\{ \begin{matrix}{R_{Pore} = {{\int_{0}^{l_{1}}\frac{{dx}_{1}}{\Sigma C_{Pore}\pi r_{1}^{2}}} = {\frac{l_{1}}{\pi \Sigma {r_{1}^{2}\left( {C_{C} - C_{I}} \right)}}{\ln \left( \frac{C_{C}}{C_{I}} \right)}}}} \\{R_{FP} = {{\int_{0}^{l_{2}}\frac{{dx}_{2}}{\Sigma C_{FP}\pi r_{2}^{2}}} = {\frac{l_{2}}{\pi \Sigma {r_{2}^{2}\left( {C_{I} - C_{T}} \right)}}{\ln \left( \frac{C_{I}}{C_{T}} \right)}}}}\end{matrix} \right. & (18)\end{matrix}$

The resistance ratio can then be determined from Expression (18) as:

$\begin{matrix}{{\frac{R_{FP}}{R_{Pore}} = \frac{\ln \left( \frac{{C_{C}l_{2}r_{1}^{2}} + {C_{T}l_{1}r_{2}^{2}}}{{C_{T}l_{2}r_{1}^{2}} + {C_{T}l_{1}r_{2}^{2}}} \right)}{\ln \left( \frac{{C_{C}l_{2}r_{1}^{2}} + {C_{C}l_{1}r_{2}^{2}}}{{C_{C}l_{2}r_{1}^{2}} + {C_{T}l_{1}r_{2}^{2}}} \right)}}.} & (19)\end{matrix}$

To obtain a maximum voltage signal, this ratio of resistance isoptimally unity, or

$\frac{R_{FP}}{R_{Pore}} = {1.}$

By setting this ratio to the value 1 in expression (19), then:

$\begin{matrix}{{\ln \left( \frac{{C_{C}l_{2}r_{1}^{2}} + {C_{C}l_{1}r_{2}^{2}}}{{C_{C}l_{2}r_{1}^{2}} + {C_{T}l_{1}r_{2}^{2}}} \right)} = {\ln \left( \frac{{C_{C}l_{2}r_{1}^{2}} + {C_{T}l_{1}r_{2}^{2}}}{{C_{T}l_{2}r_{1}^{2}} + {C_{T}l_{1}r_{2}^{2}}} \right)}} & (20)\end{matrix}$

To simplify this expression, the ratio of ionic concentrations in thecis and trans chambers, C_(C)/C_(T), can be defined as R_(C), and theratio of the fluidic passage radius to the nanopore radius, r₂/r₁, canbe defined as R_(r). The aspect ratio of the nanopore, AR_(Pore), can bedefined as AR_(Pore)=l₁/r₁, and the aspect ratio of the fluidic passage,AR_(FP), can be defined as AR_(FP)=l₂/r₂. The simplified expression isthen given as:

$\begin{matrix}{{AR_{FP}} = {\frac{1}{\sqrt{R_{C}}} \cdot R_{r} \cdot {AR}_{Pore}}} & (21)\end{matrix}$

This expression shows directly that to match the resistance of thefluidic passage with the nanopore resistance, the aspect ratio of thefluidic passage must increase proportionally to the ratio of the fluidicpassage radius and the nanopore radius. If the fluidic solution ionicconcentrations in the cis and trans chambers are equal, then the aspectratio of the fluidic passage is set only by the ratio of radii of thefluidic passage and the nanopore. If the fluidic solution ionicconcentrations are unequal, then the aspect ratio required of thefluidic passage to meet this condition is reduced by a correspondingfactor, given that the low-concentration solution is provided in, e.g.,the fluidic passage and trans chamber while the high-concentrationsolution is provided in the cis chamber. In one embodiment, a largeionic concentration ratio can be employed, in the manner describedabove, in concert with a fluidic passage design, to produce a reducedaspect-ratio requirement for the fluidic passage while maintaining theresistance matching between the fluidic passage and the nanopore.

When manufacturing or other considerations do not permit the fabricationof a fluidic passage with dimensions that produce a substantialresistance match to that of the nanopore, then the full ratio Expression(14) above can be considered, giving the relationship of all variablesin the ratio as:

$\begin{matrix}{\frac{R_{FP}}{R_{Pore}} = \frac{\ln \left( \frac{{R_{C}{AR}_{FP}} + {AR_{Pore}R_{r}}}{{AR_{FP}} + {AR_{Pore}R_{r}}} \right)}{\ln \left( \frac{{R_{C}{AR}_{FP}} + {R_{C}{AR}_{Pore}R_{r}}}{{R_{C}{AR}_{FP}} + {AR_{Pore}R_{r}}} \right)}} & (22)\end{matrix}$

Considering examples of this relationship, if the diameter of thefluidic passage is 1000 times larger than the diameter of the nanopore,e.g., 1.5 μm vs. 1.5 nm, then for a 3:1 aspect ratio of nanopore lengthto nanopore diameter, the aspect ratio of the fluidic passage is set as300:1 for fluidic passage length to fluidic passage diameter, here 450μm deep, to match the nanopore resistance when a difference in ionicconcentration of 100:1 is employed between cis and trans chambers. Thisresult is shown in the plot of FIG. 9, which shows the resistance ratiofor a 3:1 aspect-ratio nanopore, 100:1 ionic concentration difference,and assumption of fluidic passage diameter of 1000 time nanoporediameter.

Such perfect resistance matching is not required. As explained above,when the fluidic passage resistance is 10% of the nanopore resistance,the voltage signal, V_(Sig), is more than 30% of the maximum attainablesignal. For example, given a fluidic passage having a 50:1 aspect ratio,having a 75 μm length, such can provide >20% of the nanopore resistance,as shown in the plot of FIG. 9, and can result in ˜60% of the maximumvoltage signal, as shown in the plot of FIG. 10. Based on theresistance-voltage signal relationship, as shown in the FIG. 9 plot,there can be determined the fluidic passage aspect ratio that isrequired to meet a performance specification for the nanopore sensor.Thus, no particular fluidic passage aspect ratio is required so long asthe length of the passage is greater than the diameter of the passage.All that is required is the arrangement of an electrical transductionelement at some site in the nanopore sensor that can measure theelectrical potential local to the fluidic passage.

This nanopore arrangement including a fluidic passage connected betweenthe nanopore and one of the reservoirs can compensate for some of thelimitations imposed by only differing the cis and trans chamber ionicsolution concentrations. In a nanopore sensor including differing ionicconcentrations but no fluidic passage, the ionic concentration affectsthe nanopore resistance as well as cis and trans reservoir accessresistances, and in this scenario the ability to match the nanoporeresistance with one reservoir access resistance is limited, accordinglylimiting the voltage signal that can be produced. Further, fluidicaccess resistance is localized in the vicinity of the nanopore, and is afunction of distance from the nanopore, so that for electricaltransduction elements, such as an amphiphilic membrane layer, that aremobile, there can be significant signal fluctuation. The inclusion of afluidic passage between the nanopore and one reservoir enablesstructural definition of a fluidic resistance and adds an additionalcontrol parameter to the nanopore sensor. By control of both fluidicpassage aspect ratio as well as cis and trans reservoir ionic solutionconcentrations, the nanopore sensor can be tuned to optimize voltagesignal measurement.

The fluidic passage connecting a reservoir to a nanopore can beconfigured in any convenient arrangement that enables a selected aspectratio and integration with the nanopore sensor. Referring to FIG. 11,the fluidic passage 105 can be formed as a well, a trench, a channel, orother fluidic holding chamber in a structure provided for defining thenanopore. For example, the fluidic passage can be a duct, a channel, anopen-ended trench, a path, or other geometry in a structure 120 that isarranged with a support structure 14 in which the nanopore 12 isdisposed. In this example, a support structure layer 14 is disposed on asubstrate 120 for providing the nanopore 12. The fluidic passage isformed in the substrate 120. The walls 115 of the fluidic passage can beconfigured to provide a suitable fluidic passage cross-sectionalgeometry, e.g., generally circular, elliptical, round, square, or othergeometry. The fluidic passage is connected to a reservoir, e.g., thetrans reservoir, having any dimensionality; the figures represent thetrans reservoir schematically to indicate that the trans reservoir hasany dimensionality and is not in general a high-aspect ratio passagelike the fluidic passage.

Referring to FIG. 12, the fluidic passage 105 can be disposed opposite ananopore support structure. For example, a layer 125 of material can beprovided on a nanopore support structure 14, with the fluidic passage105 defined in the material layer 125. A substrate or structure 120 thatsupports the nanopore support structure 14 can be provided opposite thefluidic passage, on the opposite side of the support structure 14. Thefluidic passage configurations of FIGS. 11-12 demonstrate that thefluidic passage can be disposed on any convenient location of thenanopore sensor.

Referring to FIG. 13, the fluidic passage 105 can be provided as apopulation of pores, wells, channels, or other geometry, in a selectedarrangement. For example, the fluidic passage 105 can include a layer ofanodized aluminum oxide (AAO). AAO is a conventional material that canbe formed with very high aspect ratio holes e.g., >1000:1, having adiameter of about 100 nm. These AAO holes are arranged in aquasi-hexagonal lattice in two dimensions with a lattice constant in therange of several hundred nanometers. An aluminum oxide film can beanodized under controllable anodizing conditions, and such anodizationcan be conducted on a film having surface pre-patterning, to tune theAAO lattice constant and hole diameter. The population of AAO holes workin concert to provide an effective aspect ratio. This AAO arrangement isan example of a holey film, membrane, or other structure having apopulation of holes, wells, pathways, or other channels, that can beemployed as a fluidic passage.

In addition, any fluidic passage configuration can be filled with a gelor other porous substance, or a selected material that is disposed inthe fluidic passage to increase the fluidic resistivity of the fluidicpassage.

The fluidic passage can be configured in a vertical arrangement,horizontal arrangement, or combination of horizontal and verticalgeometries. Referring to FIG. 14, in one embodiment, there isschematically shown a fluidic passage including a vertical passagesection 134 and a horizontal passage section 136. Such arrangements canbe provided in, e.g., a surface layer 132 on a support structure 14, orcan be integrated into a substrate or other structural feature.

Referring to FIGS. 15A and 15B, the fluidic passage 105 can beconfigured in a lateral or vertical geometry of any suitable path forachieving a selected fluidic passage aspect ratio. As shown in theschematic view in FIG. 15A, a path that winds around itself, eitherlaterally or vertically, can be configured for achieving a selectedfluidic passage aspect ratio. As shown in the schematic view of FIG.15B, a serpentine path, either lateral or vertical, can be alternativelyemployed as a fluidic passage. No particular geometry is required, andthe fluidic passage can traverse multiple materials and differentsupport structure members in connecting between a reservoir and ananopore. The geometry can be formed in a layer on a support structure,substrate, or combination of elements in the sensor. The port 140 of thefluidic passage can be connected in the horizontal or vertical directionto provide fluidic communication between a reservoir of liquid and thenanopore.

These various fluidic passage configurations can in various embodimentsinclude, e.g., an AAO film having holes of about 150 nm in diameter,with a hole-to-hole distance of about 300 nm, and a film thickness ofbetween about 100 μm-1000 μm; a planar PDMS/oxide/nitride channel havinga width of between about 0.5 μm-1 μm, a depth of between about 100nm-200 nm, and a length of between about 200 μm-500 μm; a deep well in adielectric material with a well diameter of between about 0.5 μm-2 μmand a diameter of between about 20 μm-50 μm; and a silicon wafer wellhaving a diameter of between about 2 μm-6 μm and a depth of betweenabout 200 μm-300 μm. In each of these examples, the cis and trans ionicconcentrations can be the same or can be different, with a selectedionic concentration ratio, e.g., between about 50:1-1000:1 as a ratio ofcis:trans ionic concentrations.

Turning to implementation of a fluidic passage with an electricaltransduction element for making a local potential measurement in thefluidic passage, as explained above, a local electrical potentialmeasurement can be made in the nanopore sensor with any suitable deviceor circuit that accommodates the nanopore implementation. Referring toFIGS. 16A-16E, the fluidic passage configurations described just abovecan be adapted to include an electrical transduction element at the siteof the nanopore connection to the fluidic passage. The configuration ofFIG. 16A corresponds to the fluidic passage design of FIG. 11. Here, asilicon-on-insulator (SOI) wafer can be employed to form the substrate120 with a buried oxide layer (BOX) and silicon layer 152 provided atopthereof. The nanopore 12 can be formed in these layers 150, 152. Thesilicon layer 152 can be configured as a conductance channel for makinga local potential measurement at the site of the nanopore, withelectrically conducting source 154 and drain 156 regions provided formeasuring the conductance that is transduced by the silicon conductancechannel. Similarly, as shown in FIGS. 16B, a silicon layer 152 can beconfigured as a conductance channel region with source and drain regions154, 156, here in a layer 125 that defines the fluidic passage. As shownin FIG. 16C, a sensing electrode 158 or electrodes, of metal, carbonnanotubes, or other material, can be configured at the site of thenanopore 12 and connected for sensing by, e.g., a drain electrode 156.

Referring to FIGS. 16D-16E, lateral fluidic passage channels are shownin cross section with a transduction element. In the configuration ofFIG. 15D, the fluidic passage 105 is provided in a layer 158, such as anitride layer. The nitride layer 158 is provided on a support structure14 such as a silicon layer 152 from a SOI waver, with an underlying BOXlayer 150. Source and drain regions 154, 156 are provided in connectionto the silicon layer 152, patterned as a channel for transducing thelocal electrical potential at the nanopore. In the configuration shownin FIG. 16E, the fluidic passage, shown in cross-section to depict achannel such as that in FIGS. 15A-15B, is provided in a top layer 160,such as PDMS. The channel can be moulded into the top layer 160 and thenconnected to the nanopore sensor structure. A silicon layer 152 from aSOI wafer can be configured as a conductance channel, with source anddrain regions 154, 156, connected for transducing the local electricalpotential at the nanopore. Each of these configurations in FIGS. 16A-16Eenable a sensing of the local electrical potential in the fluidicpassage at nanopore.

For any of the nanopore sensor embodiments, with or without a fluidicpassage, a range of further electrical transduction elements can beemployed. For many applications, a nanowire-based FET device can be awell-suited device, but such is not required herein. The SET, QPC, lipidbilayer, or other device and nanopore implementation, whether biologicalor solid state, can be employed. Any circuit or device that enables alocal potential measurement can be employed.

In one example, a nanowire FET can be configured at the site of thenanopore as shown in FIG. 17 here shown without the fluidic passage forclarity. In this nanowire implementation 60, there is provided ananowire 62 on the support structure 14 in which is disposed thenanopore 12. The nanowire can be formed of any suitable electricallyconducting or semiconducting material, including fullerene structuresand semiconducting wires. The term “nanowire” as used herein refers toan electrical conduction channel that is characterized by a width thatis compatible with the signal decay length measured from the nanoporesite as described above. The channel width is preferably on the sameorder of magnitude as the decay length and can be larger. The nanowirecan be made from any semiconductor material that is stable in theselected reservoir solution.

FIG. 18 is a perspective view of an implementation 65 of the nanoporesensor of FIG. 6. Here is shown the nanowire 62 provided on a membranesupport structure 14 that is self-supported across its extent, like atrampoline, between a support frame at the edges, provided on a supportstructure 64 such as a substrate. The nanowire is provided on themembrane with a nanopore extending through the thickness of the nanowireand the membrane. As shown in FIGS. 17 and 18, the nanopore 12 does notextend across the width of the nanowire. There is a region of thenanowire that is unbroken along the extent of the nanopore so thatelectrical conduction is continuous along the length of the nanowire. Ametallization region or other electrically conducting region is providedat each end of the nanowire to form source (S) and drain (D) regions.With this configuration, the nanopore sensor can be configured with cisand trans reservoirs and a fluidic passage connected to one of thereservoirs, for detecting translocation of species from one reservoirthrough the nanopore to the other reservoir.

Referring also to FIGS. 19A-19B, the support structure and nanowireconfiguration can be implemented in a variety of alternativearrangements, and a support structure, such as a membrane support layer,is not required for applications in which a nanowire material is selfsupporting and can itself function as a support structure in which thenanopore is disposed. For example, as shown in FIGS. 19A-B, in agraphene-based nanopore sensor 68, there can be provided a support layer70, which in turn supports a graphene membrane 72. The graphene membrane72 is self-supported across an aperture in the support layer 70. Thegraphene membrane in turn supports a nanowire 62, with a nanopore 12extending through the thickness of the nanowire and the graphene, andthe nanowire remaining continuous along some point of the nanowire. Asshown in FIGS. 20A-20B, this arrangement can be altered, with thenanowire 72 instead disposed under the graphene layer 72, on a supportlayer 70.

In an alternative graphene-based nanopore sensor 75, as shown in FIGS.21A-21B, there can be configured a support structure, such as a supportlayer 70, on which is disposed a graphene layer 68 that functions toprovide a structure in which a nanopore 12 is configured and that itselffunctions to provide a nanowire. The graphene can be provided in anysuitable geometry that provides the requisite nanowire at the site ofthe nanopore 12. In this configuration, the graphene layer 68, due toits thickness and conductivity, senses the local electrical potential onboth sides of the nanopore, i.e., the conductance of the graphene layerchanges as a function of the local potential in both the trans and cisreservoirs. The nanopore sensor signal of a local potential measurementis therefore for this graphene-based nanopore sensor an indication ofthe difference between the cis and trans reservoir potentials.

Thus any in a very wide range of electrical transduction elements thatcan be employed to measure the electrical potential local to the fluidicpassage of the nanopore sensor. A semiconductor-based FET or othersensing device, a sensing metal electrode connected to a device such asan FET device, a graphene-based device, or other suitable transductionelement can be employed.

As demonstrated by these arrangements, the support layer, support layermembrane, nanowire, and support structure can be configured with any ina wide range of materials combinations and thicknesses. The fluidicpassage configurations described above can be integrated into any ofthese arrangements. For many applications, it can be preferred that thestructure in which the nanopore is disposed be as thin as possible, andpreferably no thicker than the extent of a species object, or objectregion to be detected. As explained above, support structure materialscan include nitrides, oxides, conductors, semiconductors, graphene,plastics, or other suitable material, which can be electricallyinsulating or electrically conducting.

As shown in FIGS. 22A-22D, for a nanowire implementation, the nanoporeis provided at the location of a nanowire 62 such that an unbroken,continuous path for electrical conduction is provided through thenanowire. The nanopore can be provided at a central region of thenanowire, as depicted in FIG. 22A, can be provided at an edge of thenanowire, as depicted in FIGS. 22B-22C, or can be provided at a sitenear to or adjacent to the nanowire, as depicted in FIG. 22D. In allcases, a continuous path for electrical conduction is provided throughthe nanowire.

In the nanopore arrangements of FIGS. 22A-22C, it is found that that thesensitivity of the nanopore region is also significantly enhancedcompared to the sensitivity of the same region prior to nanoporedrilling. This sensitivity localization can be understood by a modelaccounting for the reduction of the cross-sectional area of the nanowireas a conduction channel, assuming all other material properties, such asdoping level and mobility remain unchanged. The reduced cross-sectionalarea of the nanowire increases the resistance of the nanopore region andtherefore alleviates series resistance and signal attenuation from otherportions of the nanowire. Quantitatively, this sensitivity enhancementat the nanopore region can be obtained from the following equation for arectangular-shaped nanopore as an example:

$\begin{matrix}{{\Delta = \left( \frac{\rho_{0}L}{{p\left( {L - L_{0}} \right)} + {\rho_{0}L_{0}}} \right)^{2}}.} & (23)\end{matrix}$

Here, Δ is the sensitivity enhancement defined as the sensitivity of thedevice with a nanopore divided by the sensitivity without the nanopore,and ρ₀ and ρ are the linear resistivities of the nanowire conductionchannel with and without the nanopore, respectively. L is the totalchannel length and Lo is the channel length of the nanopore region,which for this square example is equal to the side length of thenanopore along the nanowire axial direction. For other portions ofnanowire, because all parameters remain the same but the total channelresistance is increased slightly due to the nanopore, the sensitivityshould decrease slightly after nanopore drilling. The combination ofincreased sensitivity at the nanopore region and decreased sensitivityof all other nanowire portions makes the sensitivity of a nanoporesensor enhanced, self-aligned and localized at the nanopore.

In fabrication of the nanopore sensor, both for embodiments including afluidic passage and embodiments not including a fluidic passage, firstconsidering a nanowire-based solid state nanopore sensor, ashort-channel nanowire can be preferred, and for many applications, asilicon nanowire (SiNW) can be preferred because the SiNW has beendemonstrated as an excellent electrical potential and charge sensor forsub-cellular and single-virus level signalling with remarkable stabilityin solution. To minimize signal attenuation from channel seriesresistance, the SiNW channel can be reduced, if desired, to less thanabout 200 nm by nickel solid-state diffusion. SiNWs can be fabricatedby, e.g., chemical vapor deposition, or other suitable process, anddisposed on a selected membrane, such as a nitride membrane, bysolution. For many applications, a commercially-available nitridemembrane chip can be suitably employed. Electron beam lithography oroptical lithography can be employed for producing source and drainelectrodes at ends of the nanowire. All electrodes and electricalcontacts are to be passivated with, e.g., a nitride or oxide material,and such can be accomplished after metal evaporation and before lift-offprocesses. The nanopore can be produced at a selected site by, e.g.,electron beam, or by other beam species or etching process that producesa selected nanopore dimension.

In fabrication of a graphene-based nanopore sensor including a nanowirestructure on top of the graphene membrane, like the graphene-basednanopore sensor of FIGS. 19A-19B, first a membrane, such as a nitridemembrane, is processed to form a micron-sized aperture in the membrane,e.g., by electron beam lithography or photolithography and reactive ionetching (RIE). Then a graphene sheet or piece is disposed on the nitridemembrane, covering the aperture, to form a graphene membrane. Thegraphene sheet can be synthesized by CVD or other process, or producedby mechanical exfoliation, and transferred to the nitride membrane, overthe nitride membrane aperture.

Electron beam lithography or photolithography can then be conducted withmetal evaporation to define electrodes in the conventional manner on thenitride membrane. Dielectrophoresis or other suitable process can thenbe employed to align a nanowire, such as a silicon nanowire, on top ofthe graphene membrane at the location of the aperture in the nitridemembrane. Electron beam lithography or photolithography can then beconducted with metal evaporation to define the source and drain contactsat ends of the SiNW. Thereafter, excessive graphene can be removed byelectron beam lithography or photolithography and, e.g., UV-ozonestripper, oxygen plasma, or other suitable method to remove graphenefrom regions outside the intended graphene membrane location. Finally, ananopore is produced through a site at the nanowire and the underlyinggraphene membrane by, e.g., electron beam milling, ion beam milling,etching, or other suitable process as described above.

In fabrication of a graphene-based nanopore sensor including a graphenemembrane that is on top of a nanowire FET structure, like thegraphene-based nanopore sensor of FIGS. 20A-20B, a suitable structurecan be employed for configuring the arrangement, e.g., with asilicon-on-insulator chip (SOI). In this example, an aperture is firstformed through the backside thick silicon portion of the SOI chip, e.g.,by XF₂ etching, stopping on the oxide layer, to form an oxide-siliconmembrane. Then electron beam lithography or photolithography is employedto remove the oxide layer from the SOI chip in a smaller apertureregion, producing a membrane of silicon from the thin silicon region ofthe SOI chip. This silicon membrane is then etched to form a nanowire ofsilicon, e.g., with electron beam lithography or photolithography andchemical etching or RIE. In one example, a dove-tail-shaped Si piece isformed as shown in FIG. 20B, aligned with the aperture in the oxidemembrane of the SOI chip.

Electron beam lithography or photolithography can then be conducted withmetal evaporation to define electrodes in the conventional manner on theoxide layer. Then a graphene sheet or piece is disposed on the oxidemembrane, covering the aperture, to form a graphene membrane over thesilicon nanowire. The graphene sheet can be synthesized by CVD or otherprocess, or produced by mechanical exfoliation, and transferred to theoxide membrane, over the SiNW and oxide membrane aperture. It isrecognized that because the graphene sheet is being overlaid on top ofthe patterned silicon layer, the graphene piece may not be flat. Ifleakage is a concern for this configuration, then a thin layer of, e.g.,SiO_(x) can be coated around the graphene edges to form a sealed edgecondition.

Thereafter, excessive graphene can be removed by electron beamlithography or photolithography and, e.g., UV-ozone stripper, oxygenplasma, or other suitable method to remove graphene from regions outsidethe intended graphene membrane location. Finally, a nanopore is producedthrough a site at the overlying graphene and the silicon nanowire, e.g.,by electron beam, in relation to the location of the most narrow Sigeometry.

In fabrication of a graphene-based nanopore sensor like that depicted inFIG. 21A, first a membrane, such as a nitride membrane, is processed toform a micron-sized aperture in the membrane, e.g., by electron beamlithography or photolithography and reactive ion etching (RIE). Then agraphene sheet or piece is disposed on the nitride membrane, coveringthe aperture, to form a graphene membrane. The graphene sheet can besynthesized by CVD or other process, or produced by mechanicalexfoliation, and transferred to the nitride membrane, over the nitridemembrane aperture.

Electron beam lithography or photolithography can then be conducted withmetal evaporation to define source and drain electrodes in theconventional manner on the graphene membrane. Thereafter, the grapheneis patterned in a dovetail or other selected shape by electron beamlithography or photolithography and, e.g., UV-ozone stripper, oxygenplasma, or other suitable method to produce a narrow graphene region inthe vicinity of the selected site for a nanopore. Finally, a nanopore isproduced through the graphene membrane by, e.g., electron beam.

In fabrication of a SET-based nanopore sensor like that of FIG. 1F, anysuitable membrane material, both electrically conductive andelectrically insulating, can be employed. A nitride membrane structureor other structure can be employed, such as a graphene membrane orcombination graphene-nitride membrane structure as-described above. Ifan electrically conducting membrane material is employed, it can bepreferred to coat the material with an insulating layer, such as anoxide or nitride layer, on the side of the membrane on which the SET isto be formed. Electron beam lithography and metal evaporation techniquescan then be employed to form the source and drain regions and the SETregion out of a suitable metal. A nanopore can then be formed at thelocation of the SET in the manner given above. If an insulating layer isprovided on an electrically conducting membrane material and theinsulating layer coated the length of the nanopore through the membrane,then it can be preferred to remove that insulating material from thenanopore sidewall by, e.g., HF or other suitable etching, from thebackside of the nanopore, to remove the insulator layer from thenanopore and from the adjacent vicinity of the nanopore.

In fabrication of a QPC arrangement like that of FIG. 1G with ananopore, an SOI structure can be employed, removing the thick siliconlayer in the manner described above, and then using electron beamlithography to define the top silicon layer structure in the QPCarrangement. The nanopore can then be formed through the membrane in themanner given above.

In all of these processes, there can be included one or more processsteps and additional materials to form a fluidic passage connectedbetween the nanopore and one of the fluidic reservoirs. A planar channelcan be defined by patterned etching of a nitride or oxide layer at thenanopore site, with an oxide, glass, PDMS, or other material bonded ontothe nitride or oxide layer to seal the fluidic passage channel.Alternatively, a thick material layer, such as an oxide layer, nitridelayer, PDMS layer, polymer or other layer, can be etched or drilled,e.g., by deep reactive ion etching (RIE) to define a fluidic passage.Further, as described above, an underlying support substrate, such as asilicon wafer, can be etched, e.g., by RIE, to form a fluidic passagethrough the thickness of the wafer. These example processes are notintended to be limiting and are provided as general examples oftechniques for producing nanopore sensors. Any suitable supportstructure material and device material can be employed.

The nanopore sensor fabrication processes can be tailored to accommodateany suitable nanopore structure, whether solid state, biological, orsome combination of the two. As explained above, there can be employed aprotein nanopore disposed in an aperture of a solid state supportstructure such as an FET channel material as described above. Further asdescribed above, a protein nanopore can be employed as disposed in anamphiphilic layer, or aperture in a solid state support structure at thesite of an FET channel. Any combination of materials can be employed inthe support structure that contains the nanopore.

In each of these processes, it is preferred that the dimensions of thenanopore be selected based on a selected ratio of the reservoir buffersolution concentrations, to achieve a desired electrical potentialmeasurement in the manner described above, in conjunction withconsideration for the species objects to be investigated with thenanopore sensor. The analytical expressions above can be employed todetermine an optimum nanopore size for a given species to be detected bytranslocation through the nanopore, in concert with the other nanoporesensor parameters and operation, for enabling electrical potentialmeasurement for nanopore sensing of the species.

This is particular important for maximizing the ability to distinguishbetween different species objects as nanopore translocation of theobjects is conducted. The graphene-based nanopore sensors describedabove are particularly attractive for sensing molecular species such asDNA and other biopolymer species because the graphene thickness is onthe order of a DNA base extent. But because graphene is electricallygated on both sides of the graphene by the cis and trans reservoirsolutions, and the electrical potential in the two reservoirs isopposite, the sum of electrical potentials that is indicated by thegraphene potential measurement is smaller than that indicated by theimplementation of a nanowire on one side of a membrane. But for a smallnanopore, e.g., of about 1 nm in diameter, and with a sufficiently largeratio in buffer concentration between the cis and trans reservoirs, thesum of electrical potentials that is indicated by the graphene potentialmeasurement is comparable to that of a nanowire nanopore sensor.

In general, as nucleotides or other polymer translocates through thenanopore, the rate of translocation can be controlled by a polymerbinding moiety, for a graphene-based nanopore sensor or other nanoporesupport structure material. Typically the moiety can move the polymerthrough the nanopore with or against an applied field. The moiety can bea molecular motor using for example, in the case where the moiety is anenzyme, enzymatic activity, or as a molecular brake. Where the polymeris a polynucleotide there are a number of methods for controlling therate of translocation including use of polynucleotide binding enzymes.Suitable enzymes for controlling the rate of translocation ofpolynucleotides include, but are not limited to, polymerases, helicases,exonucleases, single stranded and double stranded binding proteins, andtopoisomerases, such as gyrases. In particular the enzyme may be ahelicase or modified helicase such as disclosed by WO2013-057495 andWO2014-013260. For other polymer types, moieties that interact with thatpolymer type can be used. The polymer interacting moiety may be anydisclosed in WO-2010/086603, WO-2012/107778, and Lieberman KR et al, JAm Chem Soc. 2010;132(50):17961-72), and for voltage gated schemes, LuanB et al., Phys Rev Lett. 2010; 104(23):238103.

It is recognized that more than one polymer unit may contribute to themeasured signal during translocation of the polymer, in which case thesignal may be referred to as being k-mer dependent, a k-mer being kpolymer units of a polymer, where k is a positive integer. The extent towhich the signal is dependent upon a k-mer is dependent upon the shapeand length of the aperture and the polymer type. For example, with thetranslocation of a polynucleotide through an MspA pore, the signal maybe considered as being dependent upon 5 nucleotide bases. Alternatively,for example where the nanopore support structure is monoatomically thin,the signal may be dependent upon only a small number of polymer unitsand may even be dominated by a single polymer unit. The measured signalsmay be used to determine a sequence probability of polymer units or todetermine the presence or absence of an analyte. Suitable exemplarymethods of signal analysis are disclosed in WO2013-041878 andWO2013-121224.

EXAMPLE I Fabrication of a SiNW FET in a Nanopore Sensor

SiNWs were synthesized using an Au-nanoparticle-catalyzed chemical vapordeposition (CVD) method. 30 nm-diameter gold nanoparticles (Ted PellaInc., Redding, Calif.) were dispersed on a silicon wafer coated with a600 nm-thick layer of silicon oxide (NOVA Electronic Materials Inc.,Flower Mound, Tex.). Boron-doped p-type SiNWs were synthesized at 435°C. and 30 Torr, with 2.4 standard cubic centimeters per minute (sccm)silane as a silicon source, 3 sccm diborane (100 ppm in helium) as aboron dopant source and 10 sccm argon as the carrier gas. The nominaldoping ratio was 4000:1 (Si:B) and the growth time was 20 minutes. Theresulting SiNWs were dissolved in ethanol by gentle sonication for ˜10seconds. Then the NW solution was deposited onto a 50 nm-thick, 100μm×100 μm silicon nitride TEM membrane grid (SPI supplies, West Chester,Pa.). Electron beam lithography and evaporation of a 60 nm-thick layerof nickel were carried out to fabricate ˜1 μm spaced-apart source anddrain electrodes on the nanowire. A layer of thickness of about 75-100nm of silicon nitride was then deposited by plasma enhanced CVD (NEXXSystems, Billerica, Mass.) on the chip immediately after metalevaporation, to passivate all electrodes.

Lift-off of the mask was then carried out to produce a nanowire on anitride membrane having passivated source and drain electrodes. Thestructure was then annealed by a rapid thermal processor (HeatPulse 610,Total Fab Solutions, Tempe, Ariz.) in forming gas at 380° C. for 135seconds to shrink the nanowire channel to an extent less than about 200nm. After conductivity testing of the resulting SiNW FET, the structurewas cleaned by UV-ozone stripper (Samco International Inc., Amityville,N.Y.) at 150° C. for 25 minutes on each side. The structure was thenloaded into a field emission transmission electron microscope (TEM)(JEOL 2010, 200 kV) and a nanopore of about 9 nm or 10 nm in extent wasdrilled by through the nanowire at a selected location by convergenthigh energy electron beam into one spot for approximately 2-5 minutes.The nanopore was sited at the edge of the nanowire, as depicted in thearrangement of FIG. 22B, whereby a substantial portion of the nanowirewidth was continuous.

EXAMPLE II Sensitivity Profiling of a SiNW FET In a Nanopore Sensor

The sensitivity of the SiNW FET sensor of the nanopore sensor wascharacterized by scanning gate microscopy (SGM). A SiNW FET device wasfabricated in accordance with the method of Example I, here with ˜2 μmlong channel length to accommodate the limited spatial resolution ofSGM. SGM was performed in a Nanoscope Ma Multi-Mode AFM (DigitalInstruments Inc., Tonawanda, N.Y.) by recording the conductance of thenanowire as a function of the position of a −10 V biased conductive AFMtip (PPP-NCHPt, Nanosensors Inc., Neuchatel, SW). The AFM tip was 20 nmabove the surface during SGM recording.

Prior to formation of a nanopore at the nanowire site, an SGM profilewas produced across the nanowire. Then a nanopore was formed at the edgeof the nanowire in the arrangement depicted in FIG. 22B. With thenanopore present, the SGM profile of the nanowire was again produced.The SGM profile was determined by averaging the conductance over theapparent width (˜100 nm) of the Si NW in a perpendicular direction usingWSxM software. FIG. 23 is a plot of sensitivity, defined as conductancechange divided by AFM tip gate voltage, along the nanowire beforenanopore formation and after nanopore formation. It is clear that thesensitivity of the device is sharply localized and aligned with thenanopore. More importantly, the sensitivity of the nanopore region isalso significantly enhanced compared to the sensitivity of the sameregion prior to nanopore formation.

EXAMPLE III Cleaning and Assembly of a Nanowire-nanopore in a NanoporeSensor

The nanowire-nanopore assembly produced by the method of Example I abovewas cleaned by UV-ozone stripper (Samco International Inc.) at 150° C.for 25 minutes on each side after formation of the nanopore. Thiscleaning process is preferred to remove any possible carbon depositionon the structure. Then the structure was annealed in forming gas at 250°C.-350° C. for 30 seconds to recover the conductance of the nanowire. Afurther 25 minute room temperature UV-ozone cleaning was performed oneach side of the structure to ensure hydrophilicity of the nanopore justbefore assembly.

To assemble the nanowire-nanopore structure with fluidic reservoirs forspecies translocation through the nanopore, PDMS chambers were sonicatedfirst in DI water, then 70% ethanol and finally pure ethanol, each for˜30 minutes and then stored in pure ethanol. Just before assembly, PDMSchambers were baked in a clean glass petri dish at ˜80° C. for ˜2 hoursto remove most of the absorbed ethanol.

A printed circuit board (PCB) chip carrier was produced for makingelectrical connection to the nanopore sensor, and was cleaned byScotch-Brite (3M, St. Paul, Minn.) to remove the copper surface oxideand any contaminants such as glue. The PCB was then sonicated inisopropyl alcohol and then in 70% ethanol, each for ˜30 minutes. Goldsolution electrodes were cleaned in piranha solution for ˜1 hour justbefore assembly.

The cleaned nanowire-nanopore structure was glued into a ˜250 μm-deepcenter pit of the PCB chip carrier using Kwik-Cast (World PrecisionInstruments, Inc., Sarasota, Fla.) silicone glue, with the device sidesurface approximately flush to the surface of the rest of PCB chipcarrier. The source and drain electrical contacts of the device werewired to copper fingers on the chip carrier by wire bonding (West-BondInc., Anaheim, Calif.). The front PDMS chamber was formed of a piece ofPDMS with a ˜1.8 mm hole in the center, with a protrusion of ˜0.5 mmaround one side of the hole opening, for pressing against the nanoporemembrane surface to ensure a tight seal. The PDMS chambers weremechanically clamped onto both sides of the chip carrier and Auelectrodes were inserted through the PDMS reservoirs. The goldelectrodes function as electrical connections for biasing the PDMSchamber solutions to produce a transmembrane voltage (TMV) for drivingspecies translocation through the nanopore electrophoretically.

The trans chamber was selected as the reservoir in which potentialmeasurements would be made for the nanopore sensor. Thus, the assemblywas arranged with the membrane oriented such that the nanowire waslocated facing the trans reservoir. The trans chamber was filled with asolution having a concentration of ˜10 mM buffer, with 10 mM KCl+0.1×TAEbuffer: 4 mM tris-acetate and 0.1 mM EDTA solution. The cis chamber wasaccordingly filled with a higher ionic concentration solution to providethe requisite reservoir concentration ratio to provide a higher assessresistance at the site of local potential measurement, in the transchamber. The cis chamber was filled with a solution of ˜1 M buffer, as 1M KCl+1×TAE buffer: 40 mM tris-acetate and 1 mM EDTA. Both solutionswere auto-cleaved, degassed by house vacuum and filtered by 20 nm Anotopsyringe filter (Whatman Ltd., Kent, UK) before use.

EXAMPLE IV Nanopore Sensing of DNA Translocation Through the Nanopore

The nanowire-nanopore structure produced by the methods of the examplesabove and assembled with the solutions having buffer concentrations asprescribed by Example III was operated for sensing translocation ofspecies objects, namely, double stranded DNA molecules of 1.4 nM pUC19(dsDNA). Both the ionic current through the nanopore and the currentfrom the nanowire FET device were measured.

The ionic current was amplified by an Axon Axopatch 200B patch-clampamplifier (Molecular Devices, Inc., Sunnyvale, Calif.) with β=0.1 (1 nAconvert to 100 mV) and 2 kHz bandwidth. The nanowire FET current wasamplified by a DL 1211 current amplifier (DL Instruments) with a 10⁶magnification (1 nA convert to 1 mV) and a 0.3 ms rise time. Both thetrans-membrane voltage (TMV) and voltage between the nanowire FET sourceand drain electrodes, V_(sd), were acquired by an Axon Digidata 1440Adigitizer (Molecular Devices, Inc.). Both nanopore ionic current andnanowire FET signals were fed into a 1440A digitizer, and recorded at 5kHz by a computer. Operation of the nanopore sensor was carried out in adark Faraday cage. To avoid 60 Hz noise that could be introduced by theelectrical grounding from different instruments, the ground line wasremoved from all current amplifiers and all instruments (Amplifiers anddigitizer) and the Faraday cage and were manually grounded to thebuilding ground together.

Upon introduction of the dsDNA into the cis reservoir, intermittenttranslocation events were recorded from the nanopore ionic currentsignal channel when the TMV reached ˜2 V. For the nanowire FET signalchannel, similar events were recorded in the conductance trace withalmost perfect time correlation with the ionic current measurements.FIG. 24A includes a plot, i, of the measured ionic current through thenanopore, and a plot, ii, of the measured nanowire FET conductance for a2.0 V TMV. FIG. 24B shows a plot, i, of the measured ionic currentthrough the nanopore, and a plot, ii, of the measured nanowire FETconductance for a 2.4 V TMV. As the TMV was increased, the duration andfrequency of translocation events measured by ionic current through thenanopore and measured by nanowire FET local potential sensing decreasedand increased respectively. From the plots it is shown that the localpotential measurement sensing method perfectly tracks the sensing byconventional ionic current measurement. The local potential measurementmethod thereby enables the determination of the time of and the durationof translocation of an object through the nanopore.

To directly compare the signal amplitudes of the FET local potentialmeasurement signal and the nanopore ionic current measurement signal,the FET conductance signal of ˜200 nS and baseline of ˜24 μS wasconverted into a current by multiplying the signal by the 150 mVsource-drain voltage. This calculation indicates that for ˜2 nA ofchange in ionic current through the nanopore, with a ˜12 nA baseline,there is produced an amplification to ˜30 nA of FET current in thenanowire local potential measurement, with a ˜3.6 μA baseline.Considering that current fabrication processes are not optimized for lownoise devices and that a far higher signal-to-noise ratio has beendemonstrated for SiNWs in general, the noise and signal-to-noise ratioof the nanowire FET itself is not be the fundamental limiting factor ofthis measurement.

EXAMPLE V Local Potential Measurement Dependence on cis and transReservoir Buffer Concentration Difference

To determine the impact of different ionic concentration fluids in thecis and trans reservoirs of the nanopore sensor, a nanowire-nanoporestructure was configured following the procedure in Example III above,but here with both cis and trans chambers filled with 1 M KCl bufferinstead of solutions having differing buffer concentrations. Operationof the nanopore sensor was then conducted with dsDNA provided in thetrans reservoir, following the procedure of Example IV above, with a TMVof 0.6 V. The ionic current through the nanopore was measured, as wasthe local potential, via nanowire FET conductance in the manner ofExample IV.

FIG. 24C provides plots including plot i, of the measured ionicconductance and ii, of the measured FET conductance. As shown in theplots, translocation events were sensed by changes in ionic current whenthe TMV reached 0.5-0.6 V but the simultaneously-recorded FETconductance change was negligible at that voltage. The reservoirsolution concentration ratio is therefore understood to play animportant role in the signal generation.

Under the balanced buffer solution concentration conditions (1 M/1 M) ofthis experiment, the nanopore solution resistance contributes themajority of the resistance of the nanopore sensor; thus, almost all ofthe TMV drops across the nanopore. The electrical potential in thevicinity of the nanowire sensor is accordingly for this condition veryclose to ground regardless of any change in the solution resistance ofthe nanopore and access resistances of the reservoirs due to blockadeduring species translocation. Under the non-balanced buffer conditions(10 mM/1 M) of Example IV above, the nanopore solution resistance andthe trans chamber access resistance are comparable, while the accessresistance of cis chamber is still negligible. Any change of thesolution resistance in the nanopore and access resistance in the transreservoir causes a corresponding redistribution of TMV and thus a changein the electrical potential in the vicinity of the nanopore at the transchamber, and this potential change is what is easily detected by thelocal potential measurement of the nanopore sensor.

This example validates the discovery herein that local potentialmeasurement nanopore sensing is preferably conducted with a differencein buffer solution concentration between the reservoirs of a nanoporesensor, and that the local potential measurement is to be conducted atthe reservoir-side of the nanopore having a higher resistance, orcorrespondingly lower concentration. This condition is not applicable tonanopore sensors wherein the membrane is sufficiently thin to operate asa nanowire and to sense the potential on both sides of the membrane, asin graphene nanopore sensors in which a graphene nanowire provides anindication of a difference between potential on the two sides of ananopore. In this case, a difference in buffer solution concentration isstill preferable, but the local potential measurement is not strictlylimited to one side or the other of the nanopore, given that thenanowire measurement inherently senses the potential on both sides ofthe membrane.

Note in the experiments above in which the reservoir solutionconcentrations were equal and were unequal required differingtransmembrane voltages to initial translocation through the nanopore.For potential measurement in the trans reservoir, with equal solutionconcentrations, the electric field through the nanopore is constant, asshown in the plot of FIG. 3D. When the reservoir concentrations aredifferent, e.g., the 100:1 concentration of the examples above, then theelectric field through the nanopore is smaller on the cis reservoir-sideof the nanopore. To produce the same electric field as that obtainedwith equal reservoir solution concentrations, the transmembrane voltageis required to be increased by about 4 times. This explains the data ofthe plots of FIGS. 23 and 24.

EXAMPLE VI Multi-Channel Nanopore Sensing

Three nanowire nanopore sensors were constructed following the methodsof the examples above. The three nanopore sensors were integrated with acommon reservoir system, with a 1 M KCl buffer solution in the cischamber and a 10 mM KCl buffer in the trans chamber. A transmembranevoltage of 3 V was employed, and 1.4 nM of pUC19 DNA was provided fortranslocation through the nanopores.

FIG. 25 provides plots i-iv of total ionic current and the nanowire FETconductance of each of the three nanopore sensors, respectively, duringDNA nanopore translocation operation. As shown in the plots, continuoustranslocation events are observed in all three nanopore sensors as wellas the total ionic current channel. All nanopore sensors operatedindependently and every falling or rising edge apparent in the ioniccurrent channel can be uniquely correlated to a corresponding edge inone of the three nanopore sensors. Using the falling and rising edge ofsignals from all three nanopore sensors to reconstruct the total ioniccurrent trace, the reconstruction is nearly perfect for of all events.This nanopore operation demonstrates that a key advantage of thenanopore sensor is the large scale integration capability. Multipleindependent nanopore sensors can be implemented without need for complexmicro-fluidic systems.

EXAMPLE VII Nanopore Sensor With a Fluidic Passage

A nanowire nanopore sensor was constructed following the methods of theexamples above. Included was a 1 μm-diameter, 50 μm-long fluidic passagein a dielectric material connected between a fluidic reservoir and thenanopore. In the nanowire was disposed a protein nanopore in a lipidbilayer in an aperture of about 100 nm in the nanowire channel. Theprotein nanopore had an effective geometry of 1.5 nm in diameter and 4.5nm in length. An ionic solution of 1.6M was provided in the cisreservoir and an ionic solution of 1 mM was provided in the transreservoir. A 300 mV bias was applied across the nanopore forelectrophoretically driving ssDNA through the nanopore.

Prior to ssDNA translocation, the electrical potential of the fluidicpassage, at the bottom of the passage, was measured to be 150 mV and thevoltage across the nanopore was 150 mV. When the ssDNA translocatedthrough the nanopore, the effective nanopore diameter was 1.12 nm, andthe electrical potential at the bottom of the fluidic passage wasmeasured to be 128 mV, with the voltage across the nanopore being 172mV. The measured voltage signal corresponding to ssDNA translocation was22 mV. This signal was distributed uniformly at the bottom of thefluidic passage, at the FET sensing surface, and did not change as thebiological nanopore moved within the lipid membrane.

With these examples and the preceding description, it is demonstratedthat the nanopore sensor can provide sensing of species translocatingthrough a nanopore and can discriminate between differing objects, suchas DNA bases, as those objects translocate through the nanopore. Thenanopore sensor is not limited to sensing of a particular species orclass of species and can be employed for a wide range of applications.It is recognized that the nanopore sensor is particularly well-suitedfor sensing of biopolymer molecules that are provided for translocationthrough the nanopore. Such molecules include, e.g., nucleic acid chainssuch as DNA strands, an oligonucleotide or section of single-strandedDNA, nucleotides, nucleosides, a polypeptide or protein, amino acids ingeneral, or other biological polymer chain. There is no particularlimitation to the species object to be sensed by the nanopore sensor.With differing reservoir solution concentrations, a fluidic passageconfiguration, or some combination of these two features, it isdemonstrated that the nanopore sensor can operate with reasonablebandwidth and sensitivity for discriminating DNA bases, and thereforeenables DNA sequencing.

It is recognized, that those skilled in the art may make variousmodifications and additions to the embodiments described above withoutdeparting from the spirit and scope of the present contribution to theart. Accordingly, it is to be understood that the protection sought tobe afforded hereby should be deemed to extend to the subject matterclaims and all equivalents thereof fairly within the scope of theinvention.

I claim:
 1. A method for calibrating a nanopore sensor comprising:providing a first fluidic solution, having a first ionic concentration,in a first fluidic reservoir, said first fluidic reservoir beingdisposed in direct fluidic connection with an inlet to a nanopore;providing a second fluidic solution, having a second ionicconcentration, in a second fluidic reservoir, said second fluidicreservoir being disposed in fluidic connection with an outlet of saidnanopore through a fluidic passage having at least one fluidic sectionwith a fluidic section length greater than a fluidic section width;measuring an electrical potential local to the fluidic passage;determining a fluidic passage resistance and a nanopore resistance basedon measured fluidic passage electrical potential; comparing the fluidicpassage resistance with the nanopore resistance; adjusting at least oneof the first ionic concentration and the second ionic concentrationbased on said comparing; and carrying out the measuring, determining,comparing, and adjusting steps until the fluidic passage resistance isbetween about 0.1 times and about 10 times the nanopore resistance. 2.The method of claim 1 wherein adjusting at least one of the first ionicconcentration and the second ionic concentration comprises adjusting atleast one of the first ionic concentration and the second ionicconcentration until one of the first ionic concentration and the secondionic concentration is at least about one hundred times greater than theother of the first ionic concentration and the second ionicconcentration.
 3. The method of claim 1 wherein adjusting at least oneof the first ionic concentration and the second ionic concentrationcomprises adjusting said first ionic concentration until said firstionic concentration is at least about one hundred times greater thansaid second ionic concentration.
 4. The method of claim 1 whereinmeasuring an electrical potential local to the fluidic passage comprisesmeasuring a signal produced by at least one electrical transductionelement disposed in the nanopore sensor at a site that produces a signalindicative of electrical potential local to the fluidic passage.
 5. Themethod of claim 1 wherein measuring an electrical potential local to thefluidic passage comprises measuring a signal produced by an electricaltransduction element disposed in the fluidic passage.
 6. The method ofclaim 1 wherein measuring an electrical potential local to the fluidicpassage comprises measuring a signal produced by an electrical circuitconnected to an electrical transduction element disposed in the fluidicpassage.
 7. The method of claim 1 wherein measuring an electricalpotential local to the fluidic passage comprises measuring a signalproduced by a transistor.
 8. The method of claim 1 wherein measuring anelectrical potential local to the fluidic passage comprises measuring avoltage of a sensing element disposed in the fluidic passage.
 9. Themethod of claim 1 further comprising operating the nanopore sensor, saidoperation comprising: providing in said first fluidic solution at leastone molecule; translocating said at least one molecule from said firstfluidic solution to said second fluidic solution through said nanoporeand said fluidic passage; and measuring a translocation electricalpotential local to the fluidic passage during the translocating of saidat least one molecule.
 10. The method of claim 9 wherein translocatingsaid at least one molecule through the nanopore comprises translocatingat least one molecule selected from DNA, DNA fragments, RNA, RNAfragments, PNA, nucleotides, nucleosides, oligonucleotides, proteins,polypeptides, amino acids and polymers.
 11. The method of claim 9further comprising producing a signal indicative of said at least onemolecule translocation.
 12. The method of claim 9 further comprisingapplying between the first ionic solution and the second ionic solutionan electrical voltage operative to electrophoretically translocate saidat least one molecule through said nanopore.
 13. The method of claim 9wherein translocating at least one molecule through said nanoporecomprises translocating at least one molecule through a proteinnanopore.
 14. The method of claim 9 further comprising producing anelectrical signal indicative of said least one molecule translocation.15. The method of claim 14 further comprising processing said electricalsignal indicative of molecule translocation as a function of time todetermine duration of translocation of said at least one moleculethrough the nanopore.
 16. The method of claim 14 further comprisingprocessing said electrical signal indicative of molecule translocationas a function of time to identify said at least one molecule.
 17. Themethod of claim 9 further comprising controlling rate of moleculetranslocation with a molecular motor disposed at the nanopore.
 18. Themethod of claim 9 wherein translocating at least one molecule throughthe nanopore and the fluidic passage comprises translocating at leastone molecule through a lateral fluidic passage section and through avertical fluidic passage section of said fluidic passage.
 19. The methodof claim 9 wherein translocating at least one molecule through thenanopore and the fluidic passage comprises translocating at least onemolecule through a lateral fluidic passage section of said fluidicpassage, the lateral fluidic passage section disposed in-plane with saidnanopore in a nanopore support structure.
 20. The method of claim 9wherein translocating at least one molecule through the nanopore and thefluidic passage comprises translocating at least one molecule through atleast two different sections of the fluidic passage.